Microslide tissue sections were deparaffinized with xylene, hydrated using a diluted alcohol series, and immersed in 0.3% H2O2 in methanol to extinguish endogenous peroxidase activity. Sections were then microwaved for 15 minutes in 10 mM citrate buffer (pH 6.0) for antigen retrieval. Each section was blocked with 4% bovine serum albumin in phosphate buffered saline with 0.1% Tween 20 (PBST) for 30 minutes to reduce non-specific staining. Sections were incubated with anti–PD-L1 (1:100, Cell Marque, Rocklin, CA, USA) or anti–PD-1 (1:100, Ventana, Tucson, AZ, USA) antibodies in PBST containing 3 mg/mL goat globulin (Sigma-Aldrich, St. Louis, MO, USA) for 60 minutes at room temperature, followed by three successive washes with a buffer. The sections were then incubated with an antimouse/rabbit antibody (Envision plus, Dako, Carpinteria, CA, USA) for 30 minutes at room temperature. The chromogen used was 3,3'-diaminobenzidine (Dako). The sections were counterstained with Meyer’s hematoxylin. For positive controls, sections of human placenta and tonsil tissue were included in each staining run. Omission of the primary antibody for placenta and tonsil tissue sections was used as a negative control.
Goat globulin
Manufactured by Merck Group
Sourced in United States
Goat globulin is a purified protein fraction derived from goat blood serum. It consists of a complex mixture of immunoglobulins and other proteins. The core function of goat globulin is to serve as a reagent for various immunological and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Envision plus, by Agilent Technologies (4 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (4 mentions)
Anti fsp 1, by Merck Group (2 mentions)
Anti fap, by Abcam (2 mentions)
Anti cd68, by Agilent Technologies (2 mentions)
Anti pdgfrβ, by Abcam (2 mentions)
Anti pdgfrα, by Cell Signaling Technology (2 mentions)
Anti sma, by Merck Group (2 mentions)
Anti pd 1, by Roche (1 mentions)
Anti cd34, by Agilent Technologies (1 mentions)
Anti cd34, by Abcam (1 mentions)
Hif 1α, by BD (1 mentions)
3 3 diaminobenzidine (dab), by Solarbio (1 mentions)
Cd163, by Merck Group (1 mentions)
5 protocols using goat globulin
1
Immunohistochemical Detection of PD-L1 and PD-1
2
Immunohistochemical Profiling of Stromal Cells
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Sections on microslides were deparaffinized with xylene, hydrated using a diluted alcohol series, and immersed in 0.3% H2O2 in methanol to quench endogenous peroxidase activity. Sections were treated with TE buffer (10 mM Tris and 1 mM EDTA, pH 9.3) at 98°C for 30 min. To reduce non-specific staining, each section was blocked with 4% bovine serum albumin in PBS with 0.1% Tween 20 for 30 min. The sections were then incubated with anti- SMA (1∶100, Millipore, Billerica, MA, USA), anti-FAP (1∶100, Abcam), anti-FSP1 (1∶100, Millipore), anti-PDGFRα (1∶100, Cell Signaling Technology), anti-PDGFRβ (1∶100, Abcam), anti-CD34 (1∶100, Dako, Glostrup, Denmark) and anti-CD68 (1∶1000, Dako) in PBST containing 3 mg/ml goat globulin (Sigma, St. Louis, MO, USA) for 60 min at room temperature, followed by three successive washes with buffer. Sections were then incubated with an anti-mouse/rabbit antibody (Envision plus, Dako) for 30 min at room temperature. The chromogen used was 3,3′-diaminobenzidine (Dako). Sections were counterstained with Meyer’s hematoxylin. Omitting the primary antibody provided negative controls for immunostaining.
3
Immunohistochemical Characterization of Tumor Microenvironment
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Sections on microslides were deparaffinized with xylene, hydrated using a diluted alcohol series, and immersed in 0.3% H2O2 in methanol to quench endogenous peroxidase activity. Sections were treated with TE buffer (10 mM Tris and 1 mM EDTA, pH 9.3) at 98°C for 30 min. To reduce non-specific staining, each section was blocked with 4% bovine serum albumin in PBS with 0.1% Tween 20 for 30 min. The sections were then incubated with anti-Tenascin-C monoclonal antibody (1:100, Abcam, UK), anti- SMA (1:100, Millipore, USA), anti-FAP (1:100, Abcam, UK), anti-FSP1 (1:100, Millipore, USA), anti-PDGFRα (1:100, Cell Signaling Technology, USA), anti-PDGFRβ (1:100, Abcam, UK), anti-CD34 (1:100, Abcam, UK), HIF1α (1:100, BD, USA) and anti-CD68 (1:1000, Dako, Denmark) in PBST containing 3 mg/ml goat globulin (Sigma, St. USA) for 60 min at room temperature, followed by three successive washes with buffer. Sections were then incubated with an anti-mouse/rabbit antibody (Envision plus, Dako, Denmark) for 30 min at room temperature. The chromogen used was 3, 3’-diaminobenzidine (Dako, Denmark). Sections were counterstained with Meyer’s hematoxylin. Omitting the primary antibody provided negative controls for immunostaining.
4
Immunohistochemical Profiling of Tissue Sections
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Tissues were serially sectioned into 4 μm-thick sections, heated in an oven at 67 ºC for 20 min, and then dewaxed in alcohol and xylene. After antigen retrieval, the samples were treated with 3% H 2 O 2 .
Subsequently, the primary antibodies (BUB1, 1:50, Abcam, United States; CD3, CD4, CD8, CD20, CD56, CD68, CD163, ready-to-use, Maixin, Fujian) along with goat globulin (3 mg/mL, Sigma, Aldrich) were used to incubate the sections at 4 ºC overnight. Sections were then probed with anti-mouse/rabbit antibodies (1:100, Solarbio, Beijing) at 37 ºC for 30 min. Finally, 3, 3′-diaminobenzidine (Solarbio, Beijing) was used to stain the sections.
Wherein a score of 0 indicated < 5% positive cells; a score of 1 indicated 6-20% positive cells; a score of 2 indicated 21-50% positive cells; and a score of 3 indicated > 50% positive cells.
Subsequently, the primary antibodies (BUB1, 1:50, Abcam, United States; CD3, CD4, CD8, CD20, CD56, CD68, CD163, ready-to-use, Maixin, Fujian) along with goat globulin (3 mg/mL, Sigma, Aldrich) were used to incubate the sections at 4 ºC overnight. Sections were then probed with anti-mouse/rabbit antibodies (1:100, Solarbio, Beijing) at 37 ºC for 30 min. Finally, 3, 3′-diaminobenzidine (Solarbio, Beijing) was used to stain the sections.
Wherein a score of 0 indicated < 5% positive cells; a score of 1 indicated 6-20% positive cells; a score of 2 indicated 21-50% positive cells; and a score of 3 indicated > 50% positive cells.
5
SCARB1 Protein Expression Quantification
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Tissue microarray samples were cut into 4 um serial sections and then were placed in an oven at 67°C for 30 min, and further dewaxing in xylene and alcohol. Then tissue samples were treated with TE buffer (pH 9.3, 1 mM EDTA, and 10 mM Tris) at 98°C for 30 min. Next, tissue samples were immersed in 3% H2O2 in order to eliminate the endogenous peroxidase activity. Then samples were incubated with primary antibodies (SCARB1, 1:100, ABclonal, United States) in phosphate-buffered saline+Tween-20 (PBST) containing 3 mg/ml goat globulin (Sigma, St. Louis, MO, United States) for 60 min at room temperature (RT). Then anti-mouse/rabbit antibody (Envision plus, Dako) were used to incubated with tissue samples for 30 min at RT. Lastly, chromogenic agent 3, 3′-diaminobenzidine (Dako) was used to stain tissue samples.
We were examined and scored the IHC results. According to the positive cells’ proportion and the staining intensity, scores were assigned: [score 0], no or less than 5% positive cells; [score 1], 6–20% positive cells; [score 2], 21–50% positive cells; [score 3], more than 50% positive cells. The staining scores of 2 or 3 were considered as high expression, the staining results were scored as high and low.
We were examined and scored the IHC results. According to the positive cells’ proportion and the staining intensity, scores were assigned: [score 0], no or less than 5% positive cells; [score 1], 6–20% positive cells; [score 2], 21–50% positive cells; [score 3], more than 50% positive cells. The staining scores of 2 or 3 were considered as high expression, the staining results were scored as high and low.
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