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. Author manuscript; available in PMC: 2022 Dec 2.
Published in final edited form as: Methods Mol Biol. 2020;2077:209–224. doi: 10.1007/978-1-4939-9884-5_14

Subcellular Localization of Histidine Phosphorylated Proteins Through Indirect Immunofluorescence

Kevin Adam, Tony Hunter
PMCID: PMC9717436  NIHMSID: NIHMS1845759  PMID: 31707661

Abstract

Immunofluorescence (IF) takes advantage of biological and physical mechanisms to identify proteins in cell or tissue samples, exploiting the specificity of antibodies and stimulated fluorescence light emission. Here, we describe an immunofluorescence staining method for the identification of histidine phosphorylated proteins that uses neutral/alkaline conditions and targeted reagents to overcome the chemical lability of histidine phosphorylation. This method describes how 1- and 3-phosphohistidine (pHis) monoclonal antibodies can be used to reveal the localization of proteins containing these elusive phosphoramidate bonds in cells. Standard procedures and materials for IF staining with adherent and nonadherent cells are described.

Keywords: Immunofluorescence, Fluorescence, Antibody, Phosphohistidine, Histidine phosphorylation, Immunostaining, Microscopy

1. Introduction

Immunofluorescence staining (IF) takes advantage of both the binding specificity of antibodies against specific epitopes and the selective visibility of different wavelengths through stimulated light emission (fluorescence) to localize the epitope-containing protein within cells or tissues. This powerful combination was described for the first time by Coons et al. almost 80 years ago [1]. An additional 20 years was required to develop this method practically, and demonstrate its utility for the detection and localisation of proteins in bacteria, for viral protein studies and for diagnosis of disease [2, 3]. Nowadays, IF is well-established as a standard procedure in clinical and research applications, to study and validate the localization of a wide range of proteins both outside and inside the cell [4, 5]. Critically, several methods for cell fixation and membrane permeabilization have been developed over the years to allow the labeling of proteins in a spatial context within cellular compartments, which is essential to visualize subcellular localization [6, 7].

Whereas immunohistochemistry (IHC) tissue staining methods generally exploit a color-producing reaction using an enzyme conjugated to antibodies, IF uses fluorophore-conjugated antibodies. This feature enables two different approaches to be taken for visualization—direct and indirect immunofluorescence, each with specific advantages and disadvantages. Direct IF is a “one-step” immunostaining procedure involving a fluorophore-conjugated antibody directly targeting its antigen. Commonly used in live cell flow cytometry, direct IF is a more simple procedure and requires a shorter incubation time. In contrast, indirect IF is a “two-step” procedure in which an unlabeled primary antibody targeting the antigen is detected by fluorophore-conjugated secondary antibodies that provide signal amplification and allow the use of different fluorophores.

A third combined IF approach can be envisaged, which uses both direct and indirect approaches simultaneously. This combined method can be used, for example, when an extracellular protein is labeled before permeabilization, and then an intracellular protein is labeled after permeabilization. However, the indirect approach remains the most commonly used; as well as permitting signal amplification, the use of different fluorophores increases flexibility so that costaining can be achieved using the same primary antibodies.

Irrespective of the approach employed, the existence of numerous fluorophores with different characteristics that can be conjugated to antibodies, using distinct excitation and emission wavelengths, is a huge advantage for colocalization of different targets/antigens [8]. Particularly because IF is compatible with imaging using confocal microscopy technologies, it permits detection of a defined fluorescent emission signal in a specific focal plane with higher-resolution than IHC, and also makes it possible to combine several successive focal planes to obtain a 3D localization [9].

Histidine phosphorylation has been known since 1962, when Boyer et al. observed for the first time the presence of a phosphorylated imidazole structure from bovine liver mitochondria [10]. Since then, it has become particularly well known due to its involvement in signal transduction in, for example, bacteria, due to autophosphorylation of transmembrane histidine kinases in two-component systems (TCSs) [11]. However, because of the acid lability and thermosensitivity of this posttranslational modification, it took over 50 years to obtain the first phosphohistidine-specific antibodies, which finally became possible through the use of stable pHis analogues [12, 13]. Here, we describe standard procedures for systematic IF studies [14], in adherent and nonadherent cells, adapted to be compatible with the detection of histidine phosphorylation using 1- and 3-pHis monoclonal antibodies for an indirect immunofluorescence approach [15, 16].

2. Materials

Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ cm at 25 °C). All the reagents and buffers are prepared and stored at room temperature (RT), unless otherwise specified in the method. Diligently follow all local waste disposal regulations when disposing of waste materials. We do not add sodium azide to antibody reagents.

2.1. Preparation of Coverslips

  1. Coverslips, 15–16 mm diameter, ~170 μm thickness (see Note 1).

  2. Sterilization solution: 70% MeOH, 1% (v/v) HCl.

  3. Poly-l-lysine solution: Resuspend 1 mg of poly-l-lysine in 20 mL sterile tissue culture grade water to obtain a 50 μg/mL solution (see Note 2).

  4. Two forceps or cotton pliers: one curved extremity, one flat extremity. Use the forceps to handle and manipulate coverslips (Fig. 1).

  5. Tissue culture hood.

  6. Twelve well plates.

Fig. 1.

Fig. 1

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Manipulate the coverslips with forceps. (a) Hold the coverslips on one edge with the flat end forceps. (b) Remove the coverslips from the well by first lifting up with the curved end forceps in one hand, then pick up the coverslips with the flat end forceps in the other hand

2.2. Cell Culture and Preparation of Cells

  1. Cell culture hood.

  2. Cell incubator (see Note 3).

  3. Cells undergoing analysis.

  4. Cell culture medium appropriate for the cells being investigated.

  5. Phosphate Buffered Saline solution stock (PBS 10×): 1.37 M sodium chloride (NaCl), 27 mM potassium chloride (KCl), 8 mM sodium phosphate dibasic (Na2HPO4), 14.7 mM sodium phosphate monobasic (NaH2PO4). PBS stock solution is typically around pH 7.4. Dissolve 80 g NaCl, 2 g KCl, 1.14 g Na2HPO4, 1.76 g NaH2PO4 in 800 mL of H2O and adjust pH to 7.4 with HCl at RT. Add water to 1 L. Autoclave and store at RT (see Notes 4 and 5).

  6. PBS (pH 8.5): Add 50 mL of PBS 10× solution stock to 450 mL water. Mix and adjust pH to 8.5 with sodium hydroxide (see Note 6). Pre-cool and keep the solution at 4 °C.

  7. Cyto-centrifuge (if required for certain types of suspension cells).

2.3. Cell Fixation, Permeabilization, and Blocking

  1. PBS (pH 8.5): Add 50 mL of PBS 10× solution stock to 450 mL water. Mix and adjust pH to 8.5 with sodium hydroxide (see Note 6). Pre-cool and keep the solution at 4 °C.

  2. Paraformaldehyde (PFA) 8% (v/v), pH 8.5: Prepare in a fume/chemical hood from pre-scored ampoules of 16% (v/v) PFA under inert gas, adding PBS volume to volume (see Notes 7 and 8). Check the pH and adjust to 8.5 if needed using NaOH (see Note 6). Aliquot before storing at −20 °C (see Note 9).

  3. PBS (pH 4): Add 50 mL of PBS 10× solution stock to 450 mL water. Mix and adjust pH to 4 with formic acid (see Note 6). Keep at RT.

  4. PBS (pH 8.5), 0.1% (v/v) Triton X-100: Add 50 μL Triton X-100 to 50 mL PBS, pH 8.5. Resuspend well by vortexing at RT (see Note 10).

  5. Tris-buffered saline (TBS) (pH 8.5): 138 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 25 mM Trizma base, 0.9 mM CaCl2, 0.5 mM MgCl2. Alternatively, prepare a stock solution of 10× TBS and dilute in water.

  6. TBS with 0.1% (v/v) Tween 20 (TBST) (pH 8.5): Add 1 mL Tween 20 to 1 L TBS 1× (see Note 10). Dissolve completely at RT and adjust the pH to 8.5 with ~250 μL NaOH (10 N).

  7. TBST (pH 8.5), 10% (w/v) BSA: Resuspend 1 g of BSA in 10 mL of TBST. Vortex for a few minutes at RT, then keep at 4 °C.

  8. TBST (pH 8.5), 1% BSA: Dilute 1 mL of TBST, 10% BSA in 9 mL of TBST. Vortex and keep at 4 °C.

2.4. Immunostaining

  1. TBS with 0.1% (v/v) Tween 20 (TBST) (pH 8.5): Add 1 mL Tween 20 to 1 L TBS 1×. Dissolve completely at RT and adjust the pH to 8.5 with ~250 μL NaOH (10 N).

  2. TBST (pH 8.5), 1% BSA: Dilute 1 mL of TBST, 10% BSA in 9 mL of TBST. Vortex and keep at 4 °C.

  3. Purified anti-N1-pHis rabbit monoclonal antibody [15, 17], clone SC1–1 hybridoma purification (2.5 μg/mL final). A commercially available version of anti-N1-pHis rabbit monoclonal antibodies, clone SC1–1 (#MABS1330, Millipore, 1:100 ~5 μg/mL) can also be used.

  4. Purified anti-N3-pHis rabbit monoclonal antibody [15, 17], clone SC44–1 hybridoma purification (2.5 μg/mL final). Another commercially available clone, clone SC56–2 (#MABS1352, Millipore, 1:100 ~5 μg/mL) can also be used.

  5. Rabbit IgG for negative control (5 mg/mL in PBS—1000×) (see Note 11).

  6. Anti-rabbit Alexa Fluor 647 (2 mg/mL—1000×) (see Note 12).

  7. Anti-rabbit Alexa Fluor 488 (2 mg/mL—1000×) (see Note 12).

  8. Two forceps or cotton pliers: one curved extremity, one flat extremity.

  9. Parafilm.

  10. Benchtop centrifuge.

  11. Kimwipes.

  12. Pipette tip box (or something of a size suitable to cover the coverslips during incubation).

  13. Twelve well plates.

2.5. Nuclei Staining, Mounting and Imaging

  1. DAPI (4′,6-diamidino-2-phenylindole): Dissolve 10 mg DAPI in 1 mL water (~36 mM). Aliquot the stock solution and keep in the dark at −20 °C. We recommend preparing an intermediate dilution at 1 mg/mL, diluting 100 μL stock solution in 900 μL PBS (see Note 13). To obtain a solution of DAPI at 2.5 μg/mL, dissolve the 1 mg/mL solution 1:400 in TBST.

  2. PBS (pH 8.5): Add 50 mL of PBS 10× solution stock to 450 mL water. Mix and adjust pH to 8.5 with sodium hydroxide. Precool and keep the solution at 4 °C.

  3. Mounting media (see Notes 14 and 15).

  4. Glass slides (see Note 16).

  5. Microscope: We capture images from a super-resolution Zeiss LSM 880 rear port laser scanning confocal microscope with an Airyscan FAST module. If using other systems, a 40× objective is required as a minimum for a magnification of 400× (when used with a 10× eyepiece) essential for studying cells and cell structure. A light source with a power supply for laser excitation is required for immunofluorescence. Detectors and filters must be selected based on the emission spectra of the fluorochrome used. A computer and a microscope imaging software are necessary to acquire and analyze data.

3. Methods

It is worth noting that the concentration of the reagents and the incubation time may need to be adapted dependent on cell type and morphology.

3.1. Coating Coverslips with Poly-L-Lysine (Suspension Cells Only)

  1. Sterilize the required number of coverslips ready for use by soaking each coverslip separately in sterilization solution (70% MeOH, 1% (v/v) HCl). Once soaked, they can be kept together in this solution until use (see Note 17). Alternatively, expose the coverslips to 30 min of UV light just before use or autoclave coverslips in a closed petri dish.

  2. Allow the sterilization/storage solution to evaporate from the sterilized cover slides and/or cover glass (see Note 17).

  3. In a tissue culture hood, coat the surface of the coverslips by immersion in 50 μg/mL of poly-l-lysine for 1 h (see Note 18).

  4. Remove excess solution by aspiration and gently wash the surface of the coverslips once with sterile tissue culture grade water.

  5. Allow the coverslips to dry for 2 h in the tissue culture hood before adding cells.

3.2. Tissue Culture and Preparation of Cells

Calculate the number of cells required for the experiment, to include two negative controls that can be used to assess nonspecific background staining and the pHis signal under degradative conditions (pH 4, 90 °C) (see Note 19). The decision tree in Fig. 2 will help you to define what cell preparation technique is the best dependent on cell type.

Fig. 2.

Fig. 2

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Decision tree of cell preparation for IF. Adherence, sensitivity to detachment and morphology are used to define the optimal approach to mount cells for immunofluorescence

3.2.1. Adherent Cells

  1. Plate cells in a tissue culture hood at a density of 104–105 cells/well directly on coverslips previously positioned in the well, as shown in Fig. 1a (see Note 20).

  2. Grow cells in standard culture medium until about 50% confluency over 24 h.

3.2.2. Nonadherent Cells

  1. Resuspend ~104–105 suspension cells in 100 μL cold PBS pH 8.5 in a tissue culture hood.

  2. If cells are sensitive to turgescence (bigger roundish cells with important cytoplasmic compartment), they can be incubated on coated slides at 4 °C for 30–60 min. If suspension cells are flat with an important nuclear compartment, cytospin them onto poly-l-lysine–coated slides, using a cyto-centrifuge at 800 rpm for 2 min, then proceed immediately to cell fixation step.

3.3. Cell Fixation

  1. Place the plate with the cells on ice.

  2. Remove the media from the cells by aspiration without directly touching the coverslips or cells. Do not let the cells dry out.

  3. Wash the cells gently by addition of 1 mL cold sterile PBS, pH 8.5 and remove the solution by aspiration as step 2. Repeat once more.

  4. Remove the plate from ice.

  5. Cover the cells with PFA 8%, pH 8.5 for 15 min at RT (see Note 21). Check the pH of the new PFA solution before use as it decays to formic acid, which decreases the fixation efficiency (see Note 22). Do not exceed the 15 min incubation time as PFA is highly concentrated for “instant” fixation (see Note 23).

3.4. Cell Membrane Permeabilization and Blocking

  1. Preincubate PBS pH 8.5 and PBS pH 4 to 4 and 90 °C respectively, to evaluate pHis-specific (acid- and heat-labile) signal. The recommended negative control without primary antibodies should be treated using conservative conditions (see Note 12).

  2. After fixation, collect the PFA solution (see Note 24). Do not let the cells dry out.

  3. Wash the cells 3× with 1 mL of the appropriate PBS solution, 5 min each time: cold PBS, pH 8.5 or warm PBS, pH 4 (see Note 25).

  4. Permeabilize fixed cells by addition of 1 mL PBS (pH 8.5), 0.1% (v/v) Triton X-100 at RT for 15 min (see Note 26).

  5. Wash 2× with 1 mL of the appropriate condition for PBS solution, 5 min each time.

  6. Wash with 1 mL of TBST, 1% BSA at RT.

  7. Block with 1 mL of TBST, 10% BSA at RT for 30 min (see Note 27).

3.5. Primary and Secondary Immunostaining

  1. Dilute the required primary antibody (1- and 3-pHis mAbs; SC1–1 and SC44–1 respectively) in 0.1% TBST (pH 8.5) BSA 1% to 2.5 μg/mL (see Note 28). Just before use, centrifuge the diluted antibody for 5 min, 20,000 × g at 4 °C to remove any precipitate from the solution.

  2. Cut a suitable size piece of Parafilm (~3 × 3 cm, depending on the number and diameter of the coverslips), place on a Kimwipes prewetted with water. Drop 10–25 μL of diluted antibody directly on the Parafilm surface for each coverslip.

  3. Carefully transfer the coverslips to the Parafilm using both curved end and flat end forceps, one to easily separate the fragile slides and a second to lift the coverslip without breaking it (Fig. 1b). Be sure to turn the coverslips’ top side (with cells on its surface) onto the droplet with the diluted antibody (see Note 29).

  4. Cover the Parafilm and humid paper with a box (e.g., pipette tip box or bigger) to create a humid environment and limit solution evaporation. Incubate with the primary antibodies at RT for 60 min.

  5. Using the curved end and flat end forceps, place the coverslips into a new plate well. Be careful to turn the coverslips to keep the cells on top. Wash immediately with 1 mL of TBST. Repeat the 1 mL wash three times, for 5 min each.

  6. Dilute each secondary antibody 1/1000 in TBST, 1% (w/v) BSA to 1 μL/mL. Protect from light and centrifuge the diluted antibodies before use for 5 min at 20,000 × g at 4 °C to avoid any precipitate in the solution (see Note 30).

  7. Add 1 mL of secondary antibody solution per coverslip and incubate at RT for 60 min in the dark.

  8. Remove the solution and immediately wash cells with 1 mL of TBST at RT. Repeat the 1 mL wash three times, for 5 min each.

3.6. Nuclei Staining and Mounting

  1. Just before use dilute DAPI to ~2.5 μg/mL (1:400) in 0.1% TBST (pH 8.5) (see Note 31).

  2. Add solution to the cells on the coverslip and leave for 5 min.

  3. Remove the staining solution and wash with PBS (pH 8.5) for 5 min. Repeat 2×.

  4. Meanwhile, label the cover glass with relevant identifiable information (e.g., cell name, experimental condition, fluorochrome, date).

  5. Drop 10 μL of mounting media on the cover glass for each coverslip.

  6. Carefully transfer the coverslip on cover glass using the curved end and flat end forceps, as mentioned earlier (see Note 29). Be careful to turn the coverslip’s top side with cells onto the mounting media droplet avoiding any bubbles.

  7. Let the coverslip air-dry in the dark for 5–10 min. Store in an opaque box on a flat surface at 4 °C overnight prior to use the confocal microscopy (see Note 32).

3.7. Imaging

  1. Bring the slides to room temperature by leaving on a bench prior to scanning on a confocal microscope (Fig. 3).

  2. Follow the microscope’s instructions for more details about the use of a confocal system and its parameters.

  3. Focus the cell image using the lower magnification (20×) with light exposure prior switching to the excitation laser (see Note 33).

  4. Re-generate the cell image using a higher magnification (60/63×, oil immersion) to get a better resolution (see Note 34).

Fig. 3.

Fig. 3

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Examples of pHis immunofluorescence labeling of formaldehyde-fixed HeLa cells. All observations were made using super-resolution microscopy on a Zeiss LSM 880 rear port laser scanning confocal microscope with an Airyscan FAST module. (a, b) Two acquisitions of HeLa cells (scale bar respectively at 20 and 10 μm) labeled with both 1-pHis mAbs and 3-pHis mAbs in green (488) and DAPI in blue (objective 63×, oil index 30). (c) One acquisition of HeLa cells (scale bar at 50 μm) labeled with rabbit IgG control in green (488) and DAPI in blue (objective 20×). (d, e) Two acquisitions of HeLa cells (scale bar respectively at 20 and 10 μm) labeled with both 1-pHis mAbs and 3-pHis mAbs in red (647) and DAPI in blue (objective 63×, oil index 30)

Acknowledgments

This work was supported by the Waitt Advanced Biophotonics Core Facility of the Salk Institute with funding from NIH-NCI CCSG: P30 014195 and the Waitt Foundation.

Footnotes

1.

Round coverslips fit better in 12-well plates. A diameter of 12 mm is sufficient and small enough to limit the volume of antibody needed, whereas a 22 mm permits more cells to be mounted on the surface, increasing the choice of cellular area that can be observed. We would recommend 15–16 mm as a good compromise. The thickness of the coverslip can impact the quality and intensity of the image; 170 μm thickness is standardly compatible with all lens magnifications.

2.

Poly-l-lysine is polycationic and interacts with anionic surfaces of cell membranes to facilitate adherence. It exists in either D or L chirality: poly-l-lysine (natural form) and poly-d-lysine (artificial form). Artificial poly-d-lysine is more resistant to enzymatic degradation compared to poly-l-lysine, but a preference can be cell dependent.

3.

A standard cell culture incubator with 5–10% CO2, 37 °C and sufficient humidity is fine for mammalian cells. Naturally, these parameters need to be adapted as a function of the cell type and experimental conditions.

4.

When making solutions, the pH is measured using a pH meter. pH stability after a period of storage can be checked using pH paper.

5.

PBS is preferentially used without added Ca2+ and Mg2+ (PBS−/−), except if you are using adherent cells that are sensitive to detachment. Some pHis signaling has been related to ion channel activity. In order to limit the impact of PBS on ion channel function, we recommend avoiding the addition of Ca2+ and Mg2+ unless absolutely necessary. Should detachment of adherent cells be overserved, add 0.9 mM calcium chloride and 0.5 mM magnesium chloride to 1× PBS buffer to improve adhesion.

6.

Adjust the pH with adequate buffering solution: sodium hydroxide 10 N (NaOH) to increase the pH value and formic acid 30% to decrease the pH. A few drops can be used initially to narrow the gap from the starting pH to the required pH. From then on, it is better to use a series of NaOH (e.g., 5 and 1 N) and formic acid (e.g., 10% and 5%) with lower ionic strengths to avoid a sudden increase/decrease in pH.

7.

Paraformaldehyde used for fixation is free of methanol but complemented with glutaraldehyde and acrolein fixatives, more appropriate for preserving cellular structure and biomolecular distribution [6].

8.

Select only PFA in pre-scored amber ampules under inert gas. This is important to protect the solution from both air oxidation and light as free formaldehyde oxidizes to formic acid with oxygen.

9.

Aliquots frozen at −20 °C are stable for years. Do not re-freeze a thawed aliquot but keep at 4 °C if you plan to use it within a week. It is essential to use fresh PFA (a new aliquot or one that is less than 3–4 weeks old when stored at 4 °C) because PFA breaks down in solution (free formaldehyde oxidizes to formic acid with oxygen). It is important not to warm-up or thaw PFA at temperatures above 60 °C because it decreases the fixation efficiency.

10.

Due to the viscosity of Triton X-100 and Tween 20, it can be challenging to obtain an accurate volume. Cutting about one third off the pipette tip with scissors makes pipetting easier, as the opening of the tip is larger. To be more accurate, Triton X-100 or Tween 20 can also be weighed to obtain the correct volume: the density is 1.065 and 1.095 g/mL, respectively.

11.

Rabbit IgGs are used as a negative control, not only to check for nonspecific background but also to evaluate washing efficiency.

12.

Fixation procedures (using PFA or formalin fixatives) cause autofluorescence in the green spectrum. We recommend using a fluorophore in the near-infrared range if you have an infrared detection system and/or to have a control condition to determine nonspecific background signal without using pHis primary antibodies, but rabbit IgG and secondary only.

13.

DAPI is a known mutagen and should be handled with care. It has poor solubility in water; sonicate as necessary to dissolve. The 10 mg/mL DAPI stock solution may be stored at −20 °C for years. The intermediate diluted stock at 1 mg/mL can be stored at 4 °C for up to 6 months or at −20 °C for years.

14.

Mounting media with DAPI can be used but is optional. We recommend using a mounting media without DAPI to keep the freedom of choice of different nuclear staining strategies or to limit potential background if you do not need to stain the nucleus.

15.

Any standard mounting media can be used (e.g., Fluoromount-G from Southern Biotech, Vectashield from Vector Laboratories, Prolong Gold from Molecular Probes, Glycerol 90% (v/v), Mowiol 4–88 10% (w/v)). However, we recommend using mounting media with antifading, such as paraphenylenediamine, to maintain fluorescence and suppress quenching.

16.

We do not recommend plastic slides but instead Quartz (UV transparency) or borosilicate glass (UV absorption), optically clear, for a better thermal and corrosion resistance.

17.

Let the MeOH/HCl evaporate totally from the surface before using the coverslip/cover glass. Do not touch the coverslip with your fingers but use the forceps. Coverslips are fragile and must be manipulated with care. The sterile inside layer of the multiwell plate’s lid can be used to position the coverslips for drying in a tissue culture hood.

18.

If the coverslips are to be coated immediately prior to cell culture, coat the coverslips in the well plate designed to grow cells, then add 0.1–1 mL poly-l-lysine to cover the top side, where the cells will attach. Incubate, wash and dry the coverslips as described, then add cells in medium. If the coverslips are coated in advance, use a petri dish with 1 mL poly-l-lysine, immerse the coverslips in solution, then incubate, wash and dry as described; maintain at 4 °C.

19.

Other negative controls can be envisaged, such as incubating with a specific pHis phosphatase, incubating a nonhydrolyzable pHis analogue peptide [15], or a histidine-phosphorylated peptide (see Chapter 13 on IHC) with the primary antibody prior to using it for IF. Degradation (hydrolysis) can also be induced by boiling the coverslips for 5–10 min in 0.01 M citrate buffer (pH 3–5), which is generally used for heat-induced antigen retrieval. Citrate-based solutions are designed to break the protein cross-links; therefore, we recommend keeping the same buffer as the conservative condition-treated sample (not treated with citrate), such that only the effects of pH and temperature are being evaluated. In parallel, it is recommended that the global pHis signal is checked from the cell lines of your choice by immunoblotting prior any IF experiments (see Chapter 12).

20.

If you plan to transfect the cells and select with antibiotics, it is recommended that you start with higher cell numbers to account for transfection efficiency.

21.

If cells reveal significant autofluorescence, alternative compatible fixation methods can be used. Instead of PFA, methanol fixation can be performed by incubating cells at −20 °C for 15 min in cold 100% methanol. Methanol fixation is useful to limit pHis hydrolysis, but we do not recommend it in the first instance as it is generally used for cytoskeletal protein staining and cannot be adapted for staining lipid-associated proteins (hydrophobic bonds), or proteins localized in the nucleus or mitochondria because of a flattening effect (crenation) [18]. A photobleaching pretreatment step can also be implemented prior to immunostaining using a white phosphor light emitting diode (LED) to eliminate the natural background from tissue or due to aldehyde fixation processes [19].

22.

Alkaline conditions used to help maintain stability of pHis are also known to improve fixation, as PFA decomposes faster at basic pH releasing formaldehyde which then fixes the sample. It also has been validated for in situ hybridization and immunohistochemistry using sodium phosphate and sodium borate [20, 21].

23.

PFA results in chemical crosslinking of free amino groups which better preserves cellular architecture. Standardly, 2–4% of PFA is sufficient to fix cells, but higher concentrations (up to 25%) are used for “instant fixation.” Even if it has been reported that cross-linking is essential for proteome-wide localization studies [14], we still recommend using 8% PFA “instant fixation” to limit the over-crosslink of biomolecules because of increasing autofluorescence.

24.

Collect the PFA solution and subsequent washes in a 50 mL Falcon tube for easy disposal of the PFA waste materials according to local regulations.

25.

Alternatively, glycine 0.1 M (3.5 g/L in the appropriate PBS solution) can be used for 5 min instead of the first wash, to react with excess formaldehyde and stop fixation.

26.

Depending on the need, other detergents like saponin can be used instead of Triton X-100 to increase ER signals. However, unlike Triton-X and Tween 20 that are nonselective and create stable pores in the membrane by interacting with both protein and lipids, saponin will not permeabilize nuclear membranes as it only removes membrane cholesterol. Furthermore, saponin can be totally washed out during the staining steps and permeabilization decreases if the saponin levels are not maintained in the subsequent washing buffer [7].

27.

BSA (Bovine Serum Albumin) is typically used for blocking nonspecific binding of nonantigen molecule. Technically, milk or any serum can be used, but note that the serum has to be from a different species than the species in which the primary antibody was raised.

28.

pHis antibodies for IF were used at 2.5 μg/mL but the concentration will need to be tested and adapted as a function of the cells/tissues used. When staining for multiple proteins or phosphorylation isomers, the primary antibodies can be combined. However, note that if you plan to distinguish them, the primary antibodies have to be derived from different species and distinct from the species being studied in order to avoid nonspecific background.

29.

To limit the suction effect on coverslips and prevent the cells from drying, do not remove the previous solution. Instead, incline the plate slightly to catch sight of the edge of the coverslips and position the curved end forceps extremity. Once the coverslips hold out of the plate, absorb by capillary action the excess solution maintaining the coverslip vertically and the edge perpendicularly in contact with absorbing paper (Kimwipes) to limit extra-dilution of antibodies.

30.

Be careful to limit light exposure as much as possible, keeping the samples in the dark during incubation times to avoid bleaching the fluorochrome. Use aluminum foil or an opaque box to protect from the light.

31.

DAPI works at a concentrations range of 0.1–10 μg/mL. If the purpose is to visualize the chromatin compaction, do not exceed 1 μg/mL to distinguish chromocenters, interchromatin space, heterochromatin and euchromatin. If DAPI is used as an internal control for cell localization, a higher concentration is recommended to reduce incubation time for staining and laser intensity required for imaging, which will decrease bleaching of higher wavelength fluorophores. Hoechst (33342) can be used instead of DAPI.

32.

Alternatively, the edges of the coverslip can be sealed with a small amount of clear nail polish. After drying, the slides can be kept at −20 °C to conserve fluorescence until imaging.

33.

It is not recommended that DAPI is exposed first due to potential bleaching of signal. Always start from the higher wavelengths and decrease to short wavelengths.

34.

Mounting media not only preserves the fluorescence but also increases the refractive index, allowing for oil immersion lens to be used for high-quality pictures.

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