Nuclei Isolation Staining (NIS) Method for Imaging Chromatin-Associated Proteins in Difficult Cell Types

Abstract

Spatial distribution of chromatin-associated proteins provides invaluable information for understanding gene regulation. Conventional immunostaining is widely used for labeling chromatin-associated proteins in many cell types. However, for a subset of difficult cell types, such as differentiated human keratinocytes, achieving high-quality immunostaining for nuclear proteins remains challenging. To overcome this technical barrier, we developed the Nuclei Isolation Staining (NIS) method. In brief, NIS involves rapid isolation of nuclei from live cells, followed by fixation and staining of the nuclei directly on coverslips for subsequent high-magnification imaging. By removing the cytoplasmic contents and staining just the nuclei, this NIS method drastically improves antibody labeling efficiency for chromatin-associated proteins. In this manuscript, we describe the development and a step-by-step protocol of NIS, using differentiated human keratinocytes as an example. We also discuss other applications, based on the principle of this NIS method, for understanding cell-type and cell-state specific gene regulation.

INTRODUCTION

Chromatin-associated proteins play critical roles in defining cell-type specificity and controlling cellular activities. Although genomic and proteomic tools have been driving the discoveries of protein-DNA and protein-protein interactions, key information is still missed from these reductionist approaches. For example, what is the spatial localization/distribution pattern of DNA and proteins inside the three-dimensional nuclei in a specific cell type? And how does this spatial localization/distribution pattern vary amongst individual cells at a given time?

A widely used technique for examining protein localization in individual cells is immunostaining, which leverages antibodies to label proteins of interest in fixed biological samples. For chromatin-associated proteins, immunostaining is conventionally applied to whole cells. A permeabilization step using detergent is typically included to facilitate antibody penetration towards the interior of the nucleus (Donaldson, 1998). Although this approach can be used to image chromatin-associated proteins in many cell types, it remains challenging for a subset of difficult cell types. One example is differentiated human keratinocytes. Keratinocytes comprise more than 90% of the epidermis and provide vital barrier function to cover and protect the entire human body (Fuchs, 2007). Undifferentiated keratinocytes, located in the innermost layer (basal layer) of the epidermis, can continuously proliferate and differentiate to replenish tissue loss (Watt, 2014). This keratinocyte differentiation process involves altered expression of thousands of genes, including the expression and crosslinking of structural proteins (such as loricrin and filaggrin) and the formation of extracellular lipid-enriched lamellar membranes, providing a strong barrier for the human body (Simpson et al., 2011). Primary human keratinocytes can be isolated and expanded in vitro in the undifferentiated state using low-calcium (0.03–0.05mM) medium. A combination of high calcium (1.2–1.5mM) and confluence induces keratinocyte differentiation in vitro in the course of several days (Hennings et al., 1980; Fuchs, 1990; Poumay and Pittelkow, 1995). This keratinocyte differentiation process modelled in vitro serves as a versatile experimental platform for understanding gene regulation in tissue differentiation.

Comparing undifferentiated and differentiated human keratinocytes, we observe drastically different labeling efficiency of chromatin-associated-proteins. Fig. 1 shows RNA Polymerase II and the chromatin remodeler Brg1 as two examples. Both proteins are expressed at similar levels in undifferentiated and differentiated keratinocytes, as confirmed by western blotting (Fig. 1A). Using the paraformaldehyde (PFA) fixation method, which is frequently used for fixing nuclear proteins, differentiated keratinocytes require three to eight times longer exposure time than undifferentiated keratinocytes to acquire images with similar brightness (Fig. 1B). These images from differentiated keratinocytes also tend to have higher background. We have also attempted the methanol-acetone fixation method, which produced weaker staining signal as compared to PFA in both undifferentiated and differentiated keratinocytes (Fig. 1C). Thus, the conventional immunostaining approaches cannot be directly applied in differentiated keratinocytes to produce high-quality immunostaining of chromatin-associated proteins.

Figure 1. Staining of chromatin-associated proteins is compromised in differentiated keratinocytes.

A) Immunoblots showing protein levels of RNA Polymerase II (Pol II) and Brg1 in undifferentiated and differentiated primary human keratinocytes. Lamin A/C was used as loading control. Protein levels of Pol II and Brg1 does not drastically change during keratinocyte differentiation. B) Staining of Pol II and Brg1 in intact undifferentiated and differentiated primary human keratinocytes with PFA fixation (4% PFA, room temperature for 15 min, permeabilization with 0.5% triton x-100). Images were taken with an EVOS Auto2 microscope using 40x short-distance objective lens. The exposure time for capturing images in undifferentiated keratinocytes was set as “1x”. Same or increased exposure time, relative to the time used for acquiring the images of the undifferentiated keratinocytes, is indicated on all other images. Other parameters including light intensity and gain were consistent in all images. Scale bar is 20 μm. C) Staining of Pol II and Brg1 in undifferentiated and differentiated primary human keratinocytes with methanol:acetone fixation (50% methanol and 50% acetone, −20°C for 10 minutes, permeabilization with 0.5% triton x-100). Exposure time, relative to the time used for acquiring images in undifferentiated keratinocytes using PFA fixation, is indicated on each image. Compared with PFA, methanol:acetone fixation decreased staining efficiency for Pol II and Brg1 in both undifferentiated and differentiated keratinocytes.

To enable efficient immunostaining of chromatin-associated proteins in differentiated keratinocytes, we developed the Nuclei Isolation Staining (NIS) method (Fig. 2). In brief, nuclei are quickly isolated from differentiated or undifferentiated keratinocytes using the same buffers as well as streamlined experimental procedures. The isolated nuclei are then fixed on poly-ornithine coated coverslips. By removing the cytoplasmic contents as well as associated plasma membrane and cortex, antibodies can penetrate efficiently into the nucleus to achieve higher imaging quality.

Figure 2. Overview of the nuclei isolation staining (NIS) approach design.

The NIS approach is designed to improve immunostaining efficiency of chromatin-associated proteins, by removing the cytoplasmic contents and directly staining the nuclei of difficult cell types such as the differentiated keratinocytes.

We initially compared three approaches, which vary in the timing of the PFA fixation step, to optimize nuclei extraction and staining conditions using undifferentiated keratinocytes (Fig. 3). To ensure that the approach does not disrupt nuclear structures, we used undifferentiated keratinocytes which are expected to remain intact during the procedure. Approach #1 extracts nuclei from keratinocytes and fixes the nuclei prior to seeding onto poly-L-ornithine coated coverslips. Approach #2 fixes cells before nuclei extraction. Approach #3 extracts nuclei and seeds them onto coated coverslips before the fixation step. To test if these approaches may alter nuclear structure, we performed immunostaining to label the nuclear-envelope marker Lamin A/C and the nucleolus marker fibrillarin. Under our current resolution (40x objective with 0.75 Numerical Aperture, conventional fluorescence microscopy), these two markers appear very similar in isolated nuclei versus intact keratinocytes (Fig. 4A), suggesting that these three methods retain the overall structure of the nucleus. suggesting that these three methods retain the overall structure of the nucleus. However, the quantity of nuclei remaining on coverslips after immunostaining were different among these approaches (Fig. 4B). With the same number of nuclei counted and seeded for all three approaches, Approach #3 showed the highest number of nuclei remaining on coverslip, with an estimated retention rate (ratio of total nuclei number quantified on a coverslip vs. estimated nuclei number that can be spun to the coverslip) of 33% after seeding and multiple washing steps during the staining process. Thus, Approach #3 was selected as the most efficient method amongst the three variations that were tested and compared.

Figure 3. Schematic illustration of three alternative approaches for NIS.

Three approaches, varying in the timing of the fixation step, were initially tested to optimize the NIS method. The first approach was designed to extract nuclei and fix the nuclei before seeing onto treated coverslips. The second approach was designed to fix intact cells prior to nuclei extraction and seeding onto coverslips. The third method was designed to fix nuclei after seeding onto coverslips.

Figure 4. Comparison of three alternative NIS approaches.

A) Immunostaining of nuclei prepared with three alternative NIS approaches versus intact undifferentiated keratinocytes, using fibrillarin and lamin A/C antibodies. There is no obvious difference of lamin and nucleolus staining pattern among these conditions. B) Quantification of nuclei retention among three approaches. Same number of nuclei were seeded and fixed using these three approaches. The number of nuclei retained on treated coverslips after staining were compared. Quantification was performed by counting number of nuclei per 5 random images of two coverslips. (Error bars represent standard deviation. ****: p<0.0001, two-way ANOVA.)

We subsequently applied this Approach #3 to stain the nuclei of undifferentiated and differentiated keratinocytes. As indicated in Fig. 5, the immunostaining efficiency of both RNA polymerase II and Brg1 is comparable in isolated nuclei, but not in intact undifferentiated versus differentiated keratinocytes. With the chromatin-associated proteins that we have tested so far, NIS improves staining in differentiated keratinocytes by approximately three to eight folds. This method opens up possibilities for future characterizations that requires high-quality imaging analysis.

Figure 5. NIS improves staining of chromatin-associated proteins in differentiated keratinocytes.

Immunostaining of RNA polymerase II (Pol II) and Brg1 comparing NIS versus whole cells of undifferentiated and differentiated keratinocytes. Exposure time, relative to the time used for acquiring images in whole cells of undifferentiated keratinocytes, is indicated on each image. Light intensity and gain were consistent among all the images. UD and DF nuclei using NIS show comparable level of Pol II and Brg1 staining, although the staining efficiency is impaired in the whole cells of differentiated keratinocytes. Scale bar is 20 μm.

The detailed protocol of this NIS method (Approach #3) is described in the following sections, using primary human keratinocytes as the example. Other potential applications of this method are discussed in the commentary section.

BASIC PROTOCOL 1

Nuclei isolation from undifferentiated and differentiated human keratinocytes

Primary human keratinocytes can now be conveniently purchased from a number of sources including the American Type Culture Collection (ATCC), Sigma and Lonza. The in vitro differentiation process of these keratinocytes is induced by confluency and high calcium (1.2mM). In general, mid-differentiation is achieved on Day 3 or Day 4, and late-differentiation is achieved on Day 6 (Kretz et al., 2012; Rubin et al., 2017; Sen et al., 2008). The time point of Day 4 differentiation is used as an example in this protocol. The nuclei from both undifferentiated and differentiated keratinocytes can be rapidly isolated using a hypotonic buffer to burst the plasma membrane, releasing the nuclei (Bao et al., 2017). A quick spin (5 to 15 seconds) is sufficient to collect the nuclei at the bottom of centrifuge tubes. The nuclei are washed one more time with hypotonic buffer to remove remaining cytoplasmic contents.

Materials:

Equipment and Supplies

• Petri dish

• CO₂ incubator (e.g., New Brunswick Galaxy 170R)

• Refrigerated centrifuge (e.g., Eppendorf 5804R refrigerated centrifuge)

• Microcentrifuge (e.g., Eppendorf 5424R refrigerated centrifuge)

• 15- or 50 mL conical tubes

• Vacuum

• Water Bath (e.g., Precision 189 Series Water Bath)

• Tissue Culture Microscope (e.g., EVOS XL Cell Imaging System)

• Hemocytometer

Cells and Culture Media

• Primary Human Keratinocytes (e.g., ATCC PCS-200–010, Sigma 102–05N, Lonza 00192627)

• Keratinocyte Media (see recipe)

• High-Calcium Keratinocyte Media (see recipe)

• DMEM–Dulbecco’s Modified Eagle Medium (e.g., GIBCO 11995–065)

Other solutions

• 120 mM CaCl₂ solution (see recipe)

• TrypLE express enzyme, no phenol red (e.g., GIBCO 12604013)

• 0.25% Trypsin, phenol red (e.g., GIBCO 25200–056)

• Nuclei extraction buffer A (NEBA; see recipe)

• Nuclei extraction buffer B (NEBB; see recipe)

• NEB (equal parts of NEBA + NEBB)

  NOTE: All cell media/TrypLE/Trypsin should be warmed to 37°C in water bath before use. Cells are cultured in incubators maintained at 37°C with 5% CO₂.

  NOTE: NEBA and NEBB solutions should be pre-cooled to 4°C and kept on ice throughout the experiment.

Protocol steps—Step annotations

Keratinocyte differentiation in vitro

• 1Expand undifferentiated keratinocytes using Keratinocyte Media, keeping the confluency below 60%.
• 2When the cell number reaches the desired amount (usually > 1 million), detach the cells from the plate by adding TrypLE for 3 minutes at 37°C.
• 3Neutralize TrypLE with equal volume of DMEM.
• 4Count the number of cells using a hemocytometer (Video of this cell-counting technique can be found at: https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells, JoVE Science Education Database, 2019)
• 5Spin down cells at 450 × g for 5 minutes, at room temperature.
• 6Resuspend the cell pellet in High-Calcium Keratinocyte Media.
• 7Seed cells to 100% confluency in desired cell culture vessel, according to the cell numbers as listed in Table 1.
• 8Allow primary human keratinocytes to differentiate for multiple days, change media using High-Calcium Keratinocyte Media every day.

Table 1:

Numbers of primary human keratinocytes needed to reach 100% confluency for inducing differentiation in different cell culture plates

Vessel Type	Cells needed to reach confluency
96-well plate	0.1 × 106 per well
48-well plate	0.2 × 106 per well
24-well plate	0.4 × 106 per well
12-well plate	0.8 × 106 per well
6-well plate	1.6 × 106 per well
6-cm plate	3.2 × 106 per plate

Nuclei Isolation

• 9Collect cells by adding 4 mL (for a 10-cm plate) of TrypLE (for undifferentiated keratinocytes) or 0.25% Trypsin (for differentiated keratinocytes) for 3–5 minutes at 37°C, respectively.
• 10Neutralize TrypLE or 0.25% Trypsin with equal volume of DMEM.
• 11Place the cell suspension in a 15- or 50-mL conical vial.
• 12Count the number of cells using a hemocytometer.
• 13Centrifuge the cell suspension for 5 minutes at 450 × g, 4°C.
• 14Remove the supernatant, leaving behind the intact cell pellet and place the vial on ice.
• 15Extract nuclei by adding 200 μL of NEBA per 1 million cells to cell pellet.
• 16Add an equal volume of NEBB to the cell suspension and gently pipette up and down 5 times.

  Avoid vigorous pipetting, which can break nuclei, release DNA and cause clumping.

• 17Incubate the sample on ice for 2 minutes, check lysis under microscope. Lysis should be at least 80% at this stage. Quickly spin down after incubation.

  Use quick spin for 15 seconds (for 5 mL Eppendorf tube) or 8 seconds (for 1.5 or 2 mL Eppendorf tube).

• 18Vacuum the supernatant containing the cytoplasmic components, and resuspend the nuclei pellet in NEB (200 uL per 1 million cells).
• 19Incubate the nuclei suspension for 5 minutes on ice. Check lysis under microscope. Incubation time can be extended to allow near complete lysis. Quickly spin down to pellet the nuclei.

  Use quick spin for 15 seconds (for 5 mL Eppendorf tube) or 8 seconds (for 1.5/2 mL Eppendorf tube.

• 20Vacuum the supernatant, leaving the nuclei pellet.
• 21Resuspend the remaining nuclei pellet in NEB so that the final concentration is 1 million nuclei per mL.

BASIC PROTOCOL 2

Attaching nuclei onto treated coverslips

This portion of the protocol is designed to attach extracted nuclei onto coverslips, through coating glass coverslips with poly-L-ornithine as well as spinning nuclei onto these coated coverslips. For beginners, 12mm coverslips are recommended to be placed in the wells of a 12-well plate (well diameter: 22.1 mm). This size difference (0.3:1 ratio of surface area) between the coverslip and the well area makes it easier for picking up coverslips, usually with a pair of tweezers, during the staining process. But this generous size difference is also accompanied by 70% loss of total nuclei to the well surface not occupied by coverslip. With more experienced users, these 12mm coverslips can be placed in 24-well plates (well diameter: 15.6mm). In this case only 40% of the nuclei are estimated to be spun onto the well surface not occupied by coverslip, recovering twice as many nuclei on the coverslips as compared to using 12-well plates.

Materials:

Lab Supplies

• Glass coverslips (e.g., Fisherbrand Microscope Cover Glass 12545-80)

• 12-well plate

Solutions

• 100% ethanol

• Poly-L-ornithine solution 0.01% (e.g., Sigma-Aldrich p4957–50mL)

• NEBA (See recipe)

• NEBB (See recipe)

• NEB (equal parts NEBA + NEBB)

• 1x Phosphate buffered saline (PBS) (e.g., GIBCO 12190–144)

Protocol steps—Step annotations

Coating coverslips with Poly-L-ornithine

• 1Place one coverslip in each well of a 12-well plate and add 1 mL of 100% ethanol to each well, respectively.
• 2Wash coverslips with ethanol for 5 minutes at room temperature.
• 3Remove the ethanol and add 1 mL of deionized water to each well. Wash coverslips with deionized water twice for 5 minutes each.
• 4Remove water from wells and incubate the washed coverslips with 1 mL of poly-L- ornithine solution per well for 5–10 minutes at room temperature. These poly-L-ornithine-treated coverslips can be stored at 4°C for up to 2 weeks.

Seeding nuclei onto coated coverslips

• 5After nuclei extraction, rinse the coated coverslips with NEB.
• 6Add resuspended nuclei (0.5 million to 1 million nuclei per coverslip) directly to poly-L- ornithine coated coverslips.
• 7Centrifuge nuclei onto coverslips for 5 minutes at 450 × g, 4°C.
• 8Fix the nuclei seeded onto the coverslip with 10% formalin for 10 minutes at room temperature.
• 9Rinse the coverslip with PBS 3 times for 10 minutes each.

  Coverslips with fixed nuclei can be stored at 4°C for up to 2 weeks before staining.

BASIC PROTOCOL 3

Staining nuclei attached on coverslips

To stain nuclei, Triton X-100 is added to PBS to permeabilize the nuclear membrane. Primary antibodies are incubated with the permeabilized nuclei on coverslips overnight at 4°C. The following day, excess primary antibodies are washed away with PBS, and secondary antibodies are added to incubate for 2 hours at room temperature.

Materials:

Solutions

• Triton X-100 (e.g., Sigma-Aldrich)

• 100% Normal goat serum (e.g., Cell Signaling Technology 5425S)

• 1x Phosphate buffered saline (PBS) (e.g., GIBCO 12190–144)

• Primary antibodies for staining (e.g., Pol II)

• Secondary antibodies for staining (e.g., Goat anti-Mouse IgG, Alexa Fluor 594)

• DAPI stain (e.g., ThermoFisher NucBlue Fixed Cell Stain ReadyProbes reagent R37606)

Protocol steps—Step annotations

1. Permeabilize nuclei on coverslips by incubating with PBS + 0.5% triton X-100 for 30 minutes and room temperature.

2. Prepare blocking solution using normal goat serum (2.5%) in PBS + 0.5% triton X-100, block at room temperature for 30 minutes.

3. Dilute primary antibodies (varied dilution) in blocking solution (PBS + 2.5% normal goat serum + 0.5% triton X-100), and incubate the coverslips in primary antibody solution overnight at 4°C.

4. The following day, wash the stained coverslips 3 times by adding ~1mL of PBS + 0.5% triton X-100 directly to the wells for 10 minutes each time at room temperature with shaking.

5. Dilute secondary antibodies (varied dilution) in blocking solution (PBS + 2.5% normal goat serum + 0.5% triton X-100). Incubate coverslips in secondary antibody solution for 2 hours, in the dark at room temperature.

6. Wash coverslips 3 times with PBS + 0.5% triton X-100 for 5 minutes at room temperature.

7. Wash coverslips twice with PBS for 5 minutes at room temperature.

8. Add DAPI stain to coverslips and incubate for 5 minutes at room temperature.

   DAPI stain is made by adding 1 drop of DAPI solution to 1 mL of NEB.

9. Wash coverslips once with PBS for 5 minutes at room temperature.

   These coverslips with nuclei are ready for imaging using microscope.

REAGENTS AND SOLUTIONS

Low-Calcium Keratinocyte media (for 1L)

• 500 mL keratinocytes SFM (GIBCO 10724–011)

• 500 mL Cascade Biologics Medium 154 (GIBCO M-154–500)

• Human keratinocyte Growth supplement (GIBCO S-001–5)

• 10 mL antibiotic antibiotic-antimycotic (GIBCO 15240–062)

• 10 mL antibiotic penicillin streptomycin (GIBCO 15140–122)

• Filter media

• Store at 4°C

High-Calcium Keratinocyte Media

• Keratinocyte media

• 1.2 mM CaCl₂ (dilute 1:100 from a 120 mM stock solution)

120 mM CaCl₂ stock solution (50 mL)

• 0.665 g CaCl₂

• 50 mL deionized water

• Store at 4°C

Nuclei extraction buffer A (NEBA):

• 10 mM Hepes pH 7.4

• 1.5 mM MgCl₂

• 10 mM KCl

• Protease inhibitor (−) EDTA (Sigma-Aldrich 11836170001)

• Store at 4°C

Nuclei extraction buffer B (NEBB):

• 10 mM HEPES pH 7.4

• 1.5 mM MgCl₂

• 10 mM KCl

• 0.4% NP-40 (Anatrace Anapoe-NID-P40 2497-59-8)

• Protease inhibitor (−) EDTA (Sigma-Aldrich 11836170001)

• Store at 4°C

COMMENTARY

Background Information

Immunostaining is a widely used technique to characterize protein localization patterns in intact cells. The general approach includes fixation of intact cells and permeabilization using detergent to facilitate antibody penetration (Donaldson, 1998). The efficiency of this approach can vary among different cell types. For example, high-quality immunostaining of chromatin-associated proteins remains technically challenging in differentiated keratinocytes. The exact mechanism impairing staining of chromatin-associated proteins remains incompletely understood. One potential factor is the altered lipid content in differentiated keratinocytes (Uchida et al., 2001; Ponec et al., 1997). Methanol-acetone fixation method can dissolve lipid, and this method works reasonably well for staining cytoplasmic proteins such as keratins. Chromatin associated proteins are often better preserved by paraformaldehyde fixation, however this fixation method can also create bonds between lipids and proteins. By removing nearly all the cytoplasmic contents in differentiated keratinocytes, this NIS protocol enables efficient antibody penetration for labeling chromatin-associated proteins. We envision that this approach can be also applied for chromatin research in other difficult cell types such as adipocytes (Malide, 2008).

Critical Parameters & Troubleshooting

Protease inhibitors must be used during nuclei isolation. Be sure to resuspend nuclei pellet slowly and gently, otherwise the nuclei extraction sample may appear clumpy. Clumping typically indicates that the nuclei have been lysed by physical shearing and genomic DNA is released. If nuclei are not adhering to the coverslips, one possibility is that the coverslips are not sufficiently coated by poly-L-ornithine. Incubation of the coverslips in the poly-L-ornithine solution can be extended with an additional 10–15 minutes in this case. Additionally, nuclei should be spun down in NEB buffer to allow efficient attachment to coverslips.

Statistical Analyses

Two-way ANOVA was done using Prism to compare nuclei retention among three alternative approaches of NIS. Nuclei retention was calculated by quantifying the number of nuclei in 5 random images from two coverslips for each approach.

Understanding Results

This NIS method is designed to improve immunostaining of chromatin-associate proteins in difficult cell types. Based on the principle of NIS, we envision a number of additional applications. For a subset of proteins that function in both cytoplasm and the nucleus, such as ERK½ and HDAC6 (Plotnikov et al., 2015; Li et al., 2013), NIS can be used to specifically characterize protein localization inside the nucleus even though these proteins are predominantly localized in the cytoplasm. This NIS approach can also be modified to characterize RNA localization/distribution inside the nucleus, using single-molecule Fluorescence In Situ Hybridization (FISH). This method opens up new possibilities for studying chromatin biology and gene regulation, and it is also compatible with super-resolution imaging.

Time Considerations

Nuclei extraction and seeding should be performed consecutively, immediately after cell trypsinization. Freezing nuclei before fixation is not recommended. Nuclei extraction and seeding to coverslips takes ~30 minutes. Once seeded and fixed, the nuclei sample can be stored at 4°C in PBS for up to two weeks. Primary and secondary antibody incubation and coverslip mounting must be done consecutively. Once mounted and sealed, coverslips can be stored in the dark at 4°C for several weeks without drastic loss of fluorescent signals. Primary antibody is incubated for ~12 hours. Secondary antibody and coverslip mounting takes ~3 hours.

Significance Statement.

The human body comprises a variety of distinct cell types, but only one genome. To understand the mechanisms underlying cell-type-specific gene regulation, spatial localization of chromatin-associated proteins can provide invaluable information. Conventional immunostaining is widely used for labeling chromatin-associated proteins in many cell types. However, for a subset of specialized cell types such as differentiated keratinocytes, it remains challenging to achieve high-quality immunostaining for imaging chromatin-associated proteins. To circumvent this barrier, we developed the Nuclei Isolation Staining (NIS) method. In brief, NIS involves rapid isolation of nuclei, followed by fixation and staining of the nuclei directly on coverslips, which drastically improves labeling efficiency for chromatin-associated proteins. This method provides new opportunities for chromatin research in difficult cell types.