Paraffin sections of 4 µm in thickness were prepared from human tissues fixed in 10% neutral formalin. For detection of CD8, tissue sections were de-paraffinized in xylene, rehydrated in diluted alcohol series, blocked with 0.3% H2O2 followed by 5% normal goat serum, and incubated with anti-CD8 primary antibodies (1:200, ab4055, Abcam, Cambridge, MA, USA) at 4 °C overnight. After incubating the tissues with biotinylated anti-rabbit secondary antibody (BP-9100, Vector Labs, Burlingame, CA, USA) at room temperature for 30 min, the signal was amplified using Vectastain ABC-HRP solution (Vector Labs) and developed using Diaminobenzidine (DAB) substrate (Vector Labs). At the end, the slides were counterstained with hematoxylin.
Vectastain abc hrp solution
Manufactured by Vector Laboratories
Sourced in United States
Vectastain ABC-HRP solution is a biotin-avidin amplification system that enhances the sensitivity of immunohistochemical and other immunodetection techniques. It consists of an avidin-biotin-peroxidase complex (ABC) that binds to biotinylated secondary antibodies, amplifying the signal for enhanced visualization of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Diaminobenzidine dab substrate, by Vector Laboratories (4 mentions)
Ab4055, by Abcam (2 mentions)
Bp 9100, by Vector Laboratories (2 mentions)
Diaminobenzidine dab, by Merck Group (1 mentions)
Anti foxm1, by Cell Signaling Technology (1 mentions)
H e staining kit, by Vector Laboratories (1 mentions)
Anti ki67, by Cell Signaling Technology (1 mentions)
Ab111101, by Abcam (1 mentions)
Ab15627, by Abcam (1 mentions)
5 protocols using vectastain abc hrp solution
1
Immunohistochemical Detection of CD8+ Cells
2
Immunohistochemical Detection of CD8
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Paraffin sections (4 μm) were prepared from human tissues fixed in 10% neutral-buffered formalin. To detect CD8, tissue sections were dewaxed in xylene, rehydrated in diluted ethanol, blocked with 0.3% H2O2, then blocked with normal goat serum, and incubated with primary rabbit anti-CD8 (ab4055, Abcam, UK) at 4°C overnight. The tissues were incubated at room temperature with biotinylated goat anti-rabbit IgG (BP-9100, Vector Labs, USA) for 30 min and then treated with Vectastain ABC-HRP solution (Vector Labs, USA) for 30 min. Diaminobenzidine (DAB) (Sigma, USA)was utilized for development.
3
Histological Characterization of Mouse Tissues
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Upon fixation in 10% formalin and embedded in paraffin, mouse tissues were processed into 4-μm sections. Staining with H&E was performed using an H&E staining kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. For immunohistochemical (IHC) staining for Ki67 and FOXM1, tissue sections underwent antigen retrieval in boiling 10 mM citrate buffer (pH 6.0) for 10 min and blocked for endogenous peroxidase activity using 0.3% H2O2 for 10 min followed by non-specific binding using 5% normal goat serum for 1 h at room temperature. The tissue sections were incubated with anti-Ki67 (1:400, #9449) or anti-FOXM1 (1:600, #20459) antibody (both from Cell Signaling Technology) at 4°C overnight, followed by biotinylated secondary antibody at room temperature for 30 min, and then with Vectastain ABC-HRP solution (Vector Labs) at room temperature for 30 min, with TBST washes in between. The signal was developed using diaminobenzidine (DAB) substrate (Vector Labs), and the slides were counterstained with hematoxylin.
4
Immunohistochemical Staining of Mouse Tissues
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Mouse tissues were fixed in 10% formalin and embedded in paraffin before prepared into 4-µm sections. Upon deparaffinization in xylene and rehydration through a series of diluted alcohol, tissue sections underwent antigen retrieval in boiled 10 mM citrate buffer (pH 6.0) for 10 min. After blocking endogenous peroxidase activity with 0.3% H2O2 in PBS for 10 min and non-specific binding with 5% normal goat serum for 1 h at room temperature, the tissue sections were incubated with either anti-F4/80 (ab111101, Abcam, Cambridge, MA, USA) or anti-Ly6c (ab15627, Abcam) antibody at 4 °C overnight. Following the incubation with biotinylated secondary antibody at room temperature for 30 min, the target signal was amplified using Vectastain ABC-HRP solution (Vector Labs, Burlingame, CA, USA) according to the manufacturer’s instructions and detected using Diaminobenzidine (DAB) substrate (Vector Labs).
5
Immunohistochemical Analysis of Mouse Tissues
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The mouse tissues were fixed in 10% neutral formalin and embedded in paraffin. Serial sections of 4 µm in thickness were prepared. For IHC detection of Ki-67 and CDK2, tissue sections were de-paraffinized in xylene and rehydrated in a series of diluted alcohol. Antigen retrieval was performed in boiling 10 mM citrate buffer (pH 6.0) for 10 min. After blocking endogenous peroxidase activity with 0.3% H2O2 in PBS for 10 min and non-specific binding with 5% normal goat serum for 1 h at room temperature, the tissue sections were incubated with anti-Ki67 (ab92742), anti-CDK2 (ab32147) and anti-SMYD3 (ab187149)antibodies. Upon three washes in 0.15 M NaCl containing 0.1% v/v Triton-X-100, pH 7.6 (TBST) buffer, the sections were incubated with biotinylated secondary antibody at room temperature for 30 min, washed three times in TBST, and then incubated in Vectastain ABC—HRP solution (Vector Labs) at room temperature for 30 min, according to the manufacturer's instructions. The signal of a target protein was developed using Diaminobenzidine (DAB) substrate (Vector Labs) and the slides were counterstained with hematoxylin.
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