Multiplex Immunofluorescent Staining in Tissue Sections
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Other organizations : University of Wisconsin–Madison
Protocol cited in 15 other protocols
Variable analysis
- Fixation in 4% paraformaldehyde
- Dehydration into methanol
- Infiltration with paraffin
- Deparaffinization in xylene
- Hydration
- Boiling in 10 mM sodium citrate (pH 6.0) for 20 min
- Incubation with primary antibodies (1:250 rabbit anti-AR, 1:250 rabbit anti-CDH1, 1:50 mouse anti-KRT14, 1:250 mouse anti-TRP63) overnight at 4°C
- Incubation with fluorescent secondary antibodies (1:250 diluted) for 1 hr
- Incubation with rabbit anti-CDH1 (1:500) and mouse anti-ACTA2 (1:300) overnight at 4°C
- Immunofluorescent staining patterns of AR, CDH1, KRT14, and TRP63
- Immunofluorescent staining patterns of CDH1 and ACTA2
- Tissue sections cut to 5 μm thickness
- Washing with TBSTw solution (25 mM Tris-HCl, pH 7.5, 140 mM NaCl, 2.7 mM KCl, 0.1% Tween-20)
- Blocking non-specific binding sites for 1 hr in TBSTw containing 1% Blocking Reagent, 5% normal goat sera, and 1% bovine serum albumin fraction 5 (RGBTw)
- Use of unlabeled secondary antibodies to block antigenic sites prior to introducing the second primary antibody for sections stained with two primary antibodies from the same host species
- Counterstaining with 4′,6-diamidino-2-phenylindole dilactate
- Mounting in anti-fade media (phosphate-buffered saline containing 80% glycerol and 0.2% n-propyl gallate)
- Fixation of ISH-stained sections in 4% PFA and bleaching with TBSTw containing 6% hydrogen peroxide
- Positive control: None mentioned
- Negative control: None mentioned
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