TABLE 4.
Label | Supplier and/or formulation |
---|---|
Nucleic acid and molecular labels, cell-type and tissue-type small-molecule stains | |
DAPI | Life Technologies (no. D-1306); 0.1–10 μg/ml |
Hoechst | Cell Signaling |
NeuroTrace 530/615 Red Fluorescent Nissl Stain | Life Technologies (no. N-21482); 1:50 dilution |
SYTO 24 | Life Technologies (no. S-7559); 1:200 dilution |
DRAQ5 | Cell Signaling (no. 4084); 1:200 dilution |
Acridine Orange | Life Technologies (no. A-1301); 100 μg/ml dilution |
Lectin, DyLight 488 conjugate | Vector Laboratories (no. L-1174); 1:100 dilution |
Methylene blue | Sigma-Aldrich (no. 66720); 1 μg/ml |
Atto-565-conjugated phalloidin | Sigma-Aldrich (no. 94072); 1:100 dilution |
Alexa Fluor 647–phalloidin | Life Technologies (no. A-22287); 0.5 μM in PBS |
Primary antibodiesa | |
Mouse anti-pan-cytokeratin (AE1/AE3) antibody, Alexa Fluor 488–conjugated | EBiosciences (no. 53–9003); 1:100 dilution |
Synthetic anti-GFAP nanobody, Atto 488–conjugated | GFAP nanobody producing according to published methods194,195 1:100 dilution (Purified GFAP nanobodies were first conjugated to Atto 488, Sigma-Aldrich, diluted in dH2O to 1 mg/ml stock and then diluted 1:100 for tissue labeling) |
Chicken anti-tyrosine hydroxylase (TH) IgY | Aves Labs (no. TYH); 1:400 dilution |
Chicken anti-glial fibrillary acidic protein (GFAP) IgY | Aves Labs (no. GFAP); 1:400 dilution |
Rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) IgG | Biocare Medical (no. CP 290A); 1:200 dilution |
Rabbit anti–integrin-β4 and anti-integrin β5 IgGs | Santa Cruz Biotechnology (β4: sc-9090, β5: sc-14010); 1:200 dilution |
Rabbit anti–β-tubulin IgG | Santa Cruz Biotechnology (no. sc-9104); 1:200 dilution |
Rabbit anti-parvalbumin antibody | Abcam (ab11427); 1:200–1:400 dilution |
Mouse anti–β-spectrin II antibody | BD Biosciences (612563); 1:200 dilution |
Rabbit anti–α-adducin antibody | Abcam (ab51130); 1:200 dilution |
Goat anti–myelin basic protein antibody | Santa Cruz Biotechnology (C-16: sc-13914); 1:200 dilution |
Mouse anti–pan-axonal neurofilament SMI-312 antibody | BioLegend (SMI-312); 1:500 dilution |
Guinea pig anti-insulin antibody | DAKO (A0564); 1:500 dilution |
Goat anti-somatostatin antibody | Santa Cruz Biotechnology (sc7819); 1:500 dilution |
Secondary antibodiesb | |
Donkey anti-chicken IgY, available as Cy2c, Cy3d, Cy5e, Alexa Fluor 594f, Alexa Fluor 647g conjugates | Jackson ImmunoResearch (703-###-155), where ### = fluorophore code; 1:200 dilution |
Donkey anti-goat IgG, available as Cy2c, Cy3d, Cy5e, Alexa Fluor 594f, Alexa Fluor 647g conjugates | Jackson ImmunoResearch (703-###-155), where ### = fluorophore code; 1:200 dilution |
Donkey anti-guinea pig IgG, available as Cy2c, Cy3d, Cy5e, Alexa Fluor 594f, Alexa Fluor 647g conjugates | Jackson ImmunoResearch (703-###-155), where ### = fluorophore code; 1:200 dilution |
Donkey anti-mouse IgG, available as Cy2c, Cy3d, Cy5e, Alexa Fluor 594f, Alexa Fluor 647g conjugates | Jackson ImmunoResearch (703-###-155), where ### = fluorophore code; 1:200 dilution |
Donkey anti-rabbit IgG, available as Cy2c, Cy3d, Cy5e, Alexa Fluor 594f, Alexa Fluor 647g conjugates | Jackson ImmunoResearch (703-###-155), where ### = fluorophore code; 1:200 dilution |
Reagentsh for FISH labeling18 | |
20-mer oligo probes toward β-actin | 1 nM each of 24 Alexa Fluor 594–labeled 20-mer oligo probes toward β-actin prepared in hybridization buffer |
Slowfade Gold + DAPI | Life Technologies (no. S-36938); mounting medium for smFISH samples |
Atto fluorescent dyes that possess an NHS ester moiety (available from Sigma-Aldrich) may be conjugated to the primary antibody; this eliminates the need to perform a secondary antibody incubation when imaging tissues via fluorescence microscopy.
Cyanine dyes are traditionally better able to withstand dehydration and embedding in nonpolar, plastic media, whereas DyLight and Alexa Fluor dyes are perceived as brighter than Cyanine dyes in aqueous medium. Both seem to work well in labeling thick, cleared tissue samples.
Cy2 code: 225.
Cy3 code: 165.
Cy5 code: 175.
Alexa Fluor 594: 585.
Alexa Fluor 647N: 605.
See Supplementary Methods for protocol.