Formalin-fixed paraffin-embedded mouse spleens were cut into 10 μm sections. The tissue slides were deparaffinized, rehydrated in decreasing alcohol concentrations and incubated in 3% H2O2 for 10 min to block endogenous peroxidase activity. Antigen retrieval was performed at 98°C for 50 min in 10 mM HIER citrate buffer pH 6 (Zytomed Systems, Berlin, Germany) in the water bath. Nonspecific protein binding was blocked with 2% goat serum and 2% BSA in PBS for 30 min. The slides were then incubated with anti-STAT5 antibody (1:200, Santa Cruz Biotechnology) overnight at 4°C. After incubation for one hour with the second anti-rabbit antibody (EnVision+ System-HRP labelled Polymer, DAKO, Hamburg, Germany) the signal was visualized by incubating the slides with AEC substrate (DAKO) and the nuclei were counterstained with hematoxylin (Carl Roth, Karlsruhe, Germany). Spleen sections from mice with FLT3-ITD-driven leukemia or transplanted with mock-transduced HSPCs were used as positive and negative controls, respectively. Staining with the secondary antibody served as further negative control
Hier citrate buffer ph 6
Manufactured by Zytomed Systems
Sourced in Germany, France
HIER citrate buffer pH 6 is a heat-induced epitope retrieval (HIER) solution used in immunohistochemistry and other similar applications. It is a buffered solution with a pH of 6.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Carl Roth (1 mentions)
Anti stat5 antibody, by Santa Cruz Biotechnology (1 mentions)
Aec substrate, by Agilent Technologies (1 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
Impress kit, by Vector Laboratories (1 mentions)
Retriever 2100, by Proteogenix (1 mentions)
2 protocols using hier citrate buffer ph 6
1
Immunohistochemical Analysis of STAT5 in Mouse Spleen
2
Ovarian BRCA2 Immunohistochemistry Protocol
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Immunohistochemistry was studied as previously described (Poulain et al., 2015) (link). Fetal human ovaries were fixed overnight in 10% neutral formalin (Carlo Erba Reagents, Val de Reuil, Frane) before being dehydrated, embedded in paraffin wax and cut into 5µm sections. After dewaxing and rehydration, antigen retrieval was performed in HIER citrate buffer pH 6 (Zytomed, Diagomics, Blagnac, France) in an autoclave (Retriever 2100, Proteogenix, Mundolsheim, France). Sections were then bathed in distilled water and incubated for 15 min in 3% H2O2 at room temperature. After 30 min in 2.5% normal Horse serum (Vector laboratories, Eurobio, Les Ulis, France), primary antibody diluted in PBS was incubated for 2h at 37°C. The primary antibody used in this study was rabbit polyclonal to human BRCA2 (1:200, Abcam, Paris, France). The primary antibody was revealed using the secondary antibody anti-rabbit IgG (IMPRESS kit, Vector Laboratories, Eurobio). Peroxidase activity was visualized using VIP (Vector laboratories, Eurobio) as a substrate. Sections were counterstained with hematoxylin.
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