TABLE 4.

Antibodies and small-molecule stains validated for cell-phenotyping in PACT- and PARS-processed tissues.

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

^aAtto 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.

^bCyanine 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.

^cCy2 code: 225.

^dCy3 code: 165.

^eCy5 code: 175.

^fAlexa Fluor 594: 585.

^gAlexa Fluor 647N: 605.

^hSee Supplementary Methods for protocol.