Mayer s hematoxylin

Immunohistochemistry was performed on 4-μm–thick formalin-fixed, paraffin-embedded human gastric biopsy sections. Subsequent to deparaffinization, antigen retrieval was performed by microwaving in 10 mmol/L citric acid buffer (pH 6) for 20 minutes for chromogranin A immunohistochemistry only. Endogenous peroxidase activity and nonspecific binding were blocked at 22°C using a peroxidase block (Dako) for 5 minutes and protein block (Dako) for 30 minutes, respectively. Tissue sections were incubated with monoclonal mouse primary PAPPA2 antibody (PA257³⁹ (link)) or polyclonal rabbit primary chromogranin A antibody (Santa Cruz Biotechnology, Heidelberg, Germany) diluted in 50 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L CaCl₂, 1% BSA, pH 7.4, for 1 hour at 22°C, followed by horseradish peroxidase–conjugated goat anti-mouse or anti-rabbit immunoglobulins (Dako) for 30 minutes at 22°C, and finally developed by incubation with 3,3′-diaminobenzidine tetra hydrochloride. Immunostained tissues were counterstained with Mayer’s hematoxylin (VWR International Ltd). Separate mouse tissue sections were stained with H&E (VWR International Ltd) and scored for quantitative histologic assessment as previously described.⁴⁰ (link)

Four μm tissue sections of FFPE tissue blocks were used for IHC. After rehydration, antigen retrieval was performed by incubating the slides for 20 minutes at 98°C in 10 mM sodium citrate buffer at pH 6.0 (BMP4) or 1 mM EDTA pH 9.0 (SMAD4 and SHH). Slides were allowed to cool down and endogenous peroxidases were blocked with Peroxidase Blocking Solution (Sakura Finetek) for 10 minutes at room temperature (RT). Nonspecific sites were blocked with UltraVision Protein Block (Thermo scientific) before primary antibody incubation. All primary antibodies used were diluted in Bright Diluent (VWR) (anti-BMP2 1:200 abcam ab6285 clone 65529.111; anti-BMP4 1:200 abcam ab124715 clone EPR6211; anti-SHH 1:200 abcam ab135240 clone 5H4; anti-SMAD4 1:200 Santa Cruz Biotechnology sc-7966 clone kappa light chain) and incubation was performed over-night at 4°C. Slides were washed in phosphate buffered saline (PBS) and incubated with a BrightVision 1 step detection system and anti-rabbit/mouse secondary antibodies (VWR). The peroxidase activity was visualized using 3,3'-Diaminobenzidine (DAB) chromogen (VWR). Finally, sections were counterstained with Mayer’s hematoxylin (VWR), dehydrated and mounted. The primary antibodies were excluded in the negative controls (PBS). H&E staining was performed in order to correlate the staining pattern against the tissue architecture.

As described by Schittny and colleagues (45 (link), 68 (link)), paraffin sections were cooked in a household pressure cooker in Target Retrieval Solution (DAKO, Glostrup, Denmark) for 13 min at 2 bar, blocked with Tris-buffered saline containing 100 mg/mL casein (Sigma), and incubated overnight at 4°C with the monoclonal rat anti-mouse Ki-67 antibody (Clone Tec-3, DAKO; diluted 1:50 in antibody diluent, DAKO). Immunoreactivity was detected using the biotinylated polyclonal rabbit anti-rat antibody (DAKO; diluted 1:200 in antibody diluent, DAKO), streptavidin-biotin horseradish peroxidase complex (DAKO), and 3-amino-9-ethylcarbazole (Sigma) as a substrate. The nuclei were counterstained with Mayer’s hematoxylin (VWR, Darmstadt, Germany).