At least three 4-micrometer sections were taken from different mouse prostates for each of the genotypes surveyed. Sections were used for H&E staining and for PRR and PACE IHC. Slides were heated for 20 minutes at 60˚C, deparaffinized in xylenes and rehydrated in ethanol gradient. Antigen retrieval was performed using EDTA pH 8.0 (PRR) buffer and sodium citrate pH 6.0 (PACE4). PACE4 slides were further autoclaved in 10 mM citrate buffer pH 6 for 45 min (16 psi, 121 ˚C). To deactivate endogenous peroxidase, 3% H2O2 was used, and slides were blocked using a protein blocking reagent (Dako) for PRR and an IHC blocking buffer (Pierce) for PACE4, at room temperature. Tissue sections were incubated with primary antibodies diluted in 5% BSA in TBST overnight at 4˚C (Abcam, ab64957, 1:600; PACE4: Abcam, ab151562, 1:400) and then with biotin-conjugated secondary antibodies (Santa-Cruz) for PRR and a secondary HRP-conjugated anti-Rabbit antibody (Biorad) for PACE4. Staining was developed using DAB (Sigma-Aldrich) containing 0.015% H2O2. Counterstaining was performed with hematoxylin.
Protein blocking reagent
Manufactured by Agilent Technologies
Protein blocking reagent is a laboratory product designed to prevent non-specific binding of proteins in various analytical techniques. It is a critical component in assays where specificity and sensitivity are essential, such as Western blotting, ELISA, and immunohistochemistry. The reagent works by occupying non-specific binding sites on the target surface, thereby reducing background signals and improving the signal-to-noise ratio of the analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab64957, by Abcam (1 mentions)
Ab151562, by Abcam (1 mentions)
Biotin conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Mca1477, by Bio-Rad (1 mentions)
Cy3 tyramide, by PerkinElmer (1 mentions)
Hoechst 3342, by Thermo Fisher Scientific (1 mentions)
Tsa cy3 system, by PerkinElmer (1 mentions)
2 protocols using protein blocking reagent
1
Immunohistochemical Analysis of Prostate
2
Multi-Dimensional Immune Profiling of Colonic Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
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H&E and immune histological staining of colonic sections with Abs directed against CD3 and Foxp3 were carried out as described previously 52 . Immunofluorescence of cryo-sections was performed using the TSA Cy3 system (PerkinElmer), a fluorescence microscope (IX70;
Olympus) and primary Abs against CD11c (HL3, BD Biosciences), MPO (15484, Abcam), and F4/80 (BM8, eBioscience). In brief, cryo-sections were fixed in ice-cold acetone for 10 minutes followed by sequential incubation with methanol, avidin/biotin (Vector Laboratories), and protein blocking reagent (Dako). Slides were then incubated overnight with primary Abs.
Subsequently, the slides were incubated for 30 minutes at room temperature (RT) with biotinylated secondary Abs (Dianova). All samples were finally treated with streptavidinhorseradish peroxidase and stained with tyramide (Cy3) according to the manufacturer's instructions (Perkin Elmer). Before examination, nuclei were counterstained with Hoechst 3342 (Invitrogen). Histological staining of splenic and thymic paraffin sections were carried out using Abs directed against CD3 (1:100, Serotec, MCA1477), B220
Olympus) and primary Abs against CD11c (HL3, BD Biosciences), MPO (15484, Abcam), and F4/80 (BM8, eBioscience). In brief, cryo-sections were fixed in ice-cold acetone for 10 minutes followed by sequential incubation with methanol, avidin/biotin (Vector Laboratories), and protein blocking reagent (Dako). Slides were then incubated overnight with primary Abs.
Subsequently, the slides were incubated for 30 minutes at room temperature (RT) with biotinylated secondary Abs (Dianova). All samples were finally treated with streptavidinhorseradish peroxidase and stained with tyramide (Cy3) according to the manufacturer's instructions (Perkin Elmer). Before examination, nuclei were counterstained with Hoechst 3342 (Invitrogen). Histological staining of splenic and thymic paraffin sections were carried out using Abs directed against CD3 (1:100, Serotec, MCA1477), B220
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