For IHC analyses, biological samples were sliced in 4-μm-thick sections, deparaffinized in xylene, and rehydrated through decreasing concentrations (100%, 95%, 80%, and 70%) of ethyl alcohol, then rinsed in distilled water. Antigen retrieval was carried out using preheated target retrieval solution for 30 minutes. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in distilled water. Slides were treated with 1% BSA and 2% FBS in PBS and then incubated in a closed humid chamber overnight at 4°C with anti-RAD51C (1:1,000, Thermo Fisher Scientific, PA5-75307) or anti-ATM antibody (1:100 in case of cell lines; 1:500 in case of PDOs, Abcam ab32420). The antibody binding was detected using a polymer detection kit (GAR-HRP, Microtech) followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab). All sections were counterstained with Mayer’s Hematoxylin (Diapath, C0305) and visualized using a bright-field microscope (Leica DM750).
Gar hrp
Manufactured by Microtech
The GAR-HRP is a laboratory instrument designed for protein detection and quantification. It utilizes the horseradish peroxidase (HRP) enzyme as a detection system. The core function of the GAR-HRP is to facilitate immunoassay-based analyses, enabling researchers to accurately measure and analyze target proteins in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Eukitt, by Bio-Optica (4 mentions)
Dm750, by Leica (3 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (3 mentions)
Ab32420, by Abcam (2 mentions)
Anti rad51c, by Thermo Fisher Scientific (2 mentions)
Jbw301, by Merck Group (1 mentions)
Gam hrp, by Microtech (1 mentions)
Ab28364, by Abcam (1 mentions)
6 protocols using gar hrp
1
IHC Analysis of RAD51C and ATM
2
Histological Analysis of Genotoxic Damage in Embryos
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Embryos at the indicated developmental stage were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. Where indicated tadpoles were irradiated with 5 Gys with Faxitron before fixation. After fixation, embryos were embedded in 1% LMP agarose and then in paraffin with Diapath automatic processor (Diapath). To assess histological features hematoxylin/eosin staining (Diapath) was performed according to standard protocol and samples were mounted in Eukitt (Bio-Optica). For IHC analysis, paraffin was removed with xylene and the sections were re-hydrated in graded alcohol. Antigen retrieval was carried out using pre-heated target retrieval solution for 45 min. Tissue sections were stained with 0.3% H2O2 for 10 min at room temperature for quenching of endogenous peroxide activity, and then blocked with 0.2% Fetal Bovine Serum (FBS) in PBS supplemented with 1% BSA for 1 h and incubated for 2 h with primary antibodies. Mouse monoclonal anti-phospho-histone H2A.X (Ser139) (JBW301, Millipore, 1:500) was used for staining. Antibody binding was detected using a polymer detection kit (GAR-HRP and GAM-HRP, Microtech), followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab). All sections were counterstained with Mayer's hematoxylin and visualized using a bright-field microscope.
3
Histopathological Analysis of Breast Tumors
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Subcutaneous breast tumours were collected and fixed in 10% neutral buffered formalin and paraffin‐embedded for histopathological analyses. To assess histological features Haematoxylin/Eosin (H&E) (Diapath) staining was performed according to standard protocol and samples were mounted in Eukitt (Bio‐Optica). The necrotic area was scored visually and the viable cells identified by the presence of nuclei. Anti CD31 (1:20, Abcam ab28364) was used to analyse tumoural angiogenesis.
For immunohistochemistry (IHC) analysis, paraffin was removed with xylene and the sections were rehydrated in graded alcohol. Antigen retrieval was carried out using a preheated target retrieval solution for 35 min. Tissue sections were blocked with FBS serum in PBS for 60 min and incubated overnight with primary antibody. The antibody binding was detected using a polymer detection kit (GAR‐HRP, Microtech) followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK‐4100; Vector Lab). All sections were counterstained with Mayer's haematoxylin and visualized using a bright‐field microscope. Samples were analysed to count the CD31‐immunopositive areas.
4
Immunohistochemical Analysis of Mouse Tissues
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Mouse tissues were fixed in 10% buffered formalin and paraffin embedded with Diapath automatic processor. To assess histological features Hematoxylin/Eosin (Diapath) staining was performed according to standard protocol and samples were mounted in Eukitt (Bio-Optica).
For immunohistochemical analysis, paraffin was removed with xylene and the sections were rehydrated in graded alcohol. Antigen retrieval was carried out using preheated target retrieval solution (pH 9.0) for 30 minutes. Tissue sections were blocked with FBS serum in PBS for 90 min and incubated overnight with primary antibodies.
For melanoma tumor antibody binding was detected using the conjugated goat anti-rabbit polymer alkaline phosphatase (AP) (Biocare) followed by a Vulcan red chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab).
For lung tumor antibody binding was detected using a polymer detection kit (GAR-HRP, Microtech) followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab). All sections were counterstained with Mayer’s hematoxylin and visualized using a bright-field microscope (LEICA DM750)
For B220 antibody binding was detected using a ABC kit Vectastain (vector, Pk4100) followed by a diaminobenzidine or Magenta chromogen (ENvision flex hrp magenta substrate chromogen system,dako) reaction.
For immunohistochemical analysis, paraffin was removed with xylene and the sections were rehydrated in graded alcohol. Antigen retrieval was carried out using preheated target retrieval solution (pH 9.0) for 30 minutes. Tissue sections were blocked with FBS serum in PBS for 90 min and incubated overnight with primary antibodies.
For melanoma tumor antibody binding was detected using the conjugated goat anti-rabbit polymer alkaline phosphatase (AP) (Biocare) followed by a Vulcan red chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab).
For lung tumor antibody binding was detected using a polymer detection kit (GAR-HRP, Microtech) followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab). All sections were counterstained with Mayer’s hematoxylin and visualized using a bright-field microscope (LEICA DM750)
For B220 antibody binding was detected using a ABC kit Vectastain (vector, Pk4100) followed by a diaminobenzidine or Magenta chromogen (ENvision flex hrp magenta substrate chromogen system,dako) reaction.
5
IHC Analysis of RAD51C and ATM
6
Immunohistochemical Analysis of Ki67 in FFPE Tissues
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Formalin-fixed paraffin-embedded tissues were sliced into serial 8-µm-thick sections and collected for immunohistochemical (IHC) staining. Human paraffin samples were stained for Haematoxylin/Eosin (Diapath) to assess histological features, according to standard protocol. For Ki67 immunoanalysis paraffin was removed with xylene and sections were rehydrated in graded alcohol. Tissue slides were incubated in 10% peroxidase solution for 1 h at 65 °C to remove melanin pigments and then antigen retrieval was carried out using preheated target retrieval solution for 45 min at 95 °C. Tissue sections were blocked with FBS serum in PBS for 60 min and incubated overnight with primary antibody (Thermoscientific, 1:50). The antibody binding was detected using a polymer detection kit (GAR-HRP, Microtech) followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab).
All sections were counterstained with Mayer's hematoxylin, mounted in Eukitt (Bio-Optica) and then visualized with an Olympus BX51 or an Olympus BX63 upright widefield microscope were normalized on nuclei and T-test statistical analysis was performed to estimate the differences between primary (N=6) and metastatic tissues (N=6). All the analysis was done in technical duplicates.
All sections were counterstained with Mayer's hematoxylin, mounted in Eukitt (Bio-Optica) and then visualized with an Olympus BX51 or an Olympus BX63 upright widefield microscope were normalized on nuclei and T-test statistical analysis was performed to estimate the differences between primary (N=6) and metastatic tissues (N=6). All the analysis was done in technical duplicates.
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