Mouse 4T1 tumor sections were deparaffinized by deparaffinization buffer (Solarbio life science). Improved Citrate Antigen Retrieval Solution (Beyotime Biotechnology) and endogenous peroxidase blocking buffer (Beyotime Biotechnology) were used for antigen retrieval and elimination of endogenous peroxidase activity, respectively. Then, the sections were blocked with goat serum in PBS for 30 min followed by incubating with antibodies at 4 °C overnight. After washing three times with PBS, tissues were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and DAB (3,3′-diaminobenzidine) HRP Color Development Kit was used for visualization. The commercially purchased antibodies used for IHC staining are shown in Table 3 .
Endogenous peroxidase blocking buffer
Manufactured by Beyotime
Sourced in China
Endogenous peroxidase blocking buffer is a solution used in immunohistochemistry and other related techniques to prevent the interference of endogenous peroxidase activity in tissue samples. It helps to minimize the background staining caused by the presence of peroxidase enzymes within the sample.
Automatically generated - may contain errors
Lab products found in correlation
Improved citrate antigen retrieval solution, by Beyotime (2 mentions)
Deparaffinization buffer, by Solarbio (1 mentions)
Rm2255, by Leica (1 mentions)
Citrate edta antigen retrieval solution, by Beyotime (1 mentions)
Quickblock, by Beyotime (1 mentions)
Diaminobenzidine dab kit, by Maixin Group (1 mentions)
Hrp labeled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Immunostaining permeabilization buffer with triton x 100, by Beyotime (1 mentions)
Anti ldha antibody, by Cell Signaling Technology (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Df2789, by Affinity Biosciences (1 mentions)
Ab138492, by Abcam (1 mentions)
Dab stain, by Beyotime (1 mentions)
Ab6586, by Abcam (1 mentions)
Biotinylated secondary antibody, by Abcam (1 mentions)
Tnf α, by Santa Cruz Biotechnology (1 mentions)
Pannoramic midi, by 3DHISTECH (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Gata4, by Abcam (1 mentions)
Citrate antigen retrieval solution, by Beyotime (1 mentions)
9 protocols using endogenous peroxidase blocking buffer
1
Immunohistochemical Staining of Mouse 4T1 Tumor Sections
2
Embryonic Head Development Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MycnOsr2 (Mycnfl/fl; Osr2IresCre) and control (Mycnfl/fl or Mycnfl/+; Osr2IresCre) mice were harvested at E13.5-E18.5 and P0. Control and mutant embryonic heads were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight, and mandibles were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 1–2 weeks. Samples were dehydrated in serial concentrations of ethanol, embedded in paraffin and sectioned at 5 μm using a microtome (RM2255; Leica Biosystems Inc., USA). For histologic analysis, deparaffinized sections were stained with haematoxylin and eosin (H&E) using standard procedures. Antigen retrieval was achieved by boiling sections in Citrate-EDTA Antigen Retrieval Solution (Beyotime Biotechnology, Shanghai, China) for 20 min in a microwave. To eliminate nonspecific binding, slides were incubated in endogenous peroxidase blocking buffer (Beyotime Biotechnology) for 20 min and blocked with QuickBlock™ (Beyotime Biotechnology) for 15 min at room temperature. Primary antibodies were incubated at 4 °C overnight. Slides were washed three times in phosphate-buffered saline (PBS) before incubation with secondary antibodies for 1 h at 37 °C. For immunohistochemical examination, immune complexes were visualized using a diaminobenzidine (DAB) kit (Maixin, Fuzhou, China).
3
Immunohistochemistry of Human Pancreatic Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human pancreatic tissue samples were collected from patients of RuiJin Hospital, Shanghai Jiaotong University, and the experimental protocols have been approved by the Ethics Committee of RuiJin Hospital, Shanghai Jiaotong University (Approval number: 2021–194). Tissues were fixed with 4% paraformaldehyde, then embedded and sliced in paraffin. The sections were dewaxed and heated in the Improved Citrate Antigen Retrieval Solution (Beyotime Biotechnology, P0083) for antigen repair and permeabilized with Immunostaining Permeabilization Buffer with Triton X-100(Beyotime Biotechnology, P0096). Endogenous peroxidase was blocked with Endogenous Peroxidase Blocking Buffer (Beyotime Biotechnology, P0100A). The sections were incubated with mouse anti-CD163 antibody (Cell Signaling Technology, 1:400) or anti-LDHA antibody (Cell Signaling Technology, 1:400). Antibody binding was detected with HRP-labeled secondary antibody (Santa Cruz Biotechnology Inc.) and visualized by the DAB+ Substrate Chromogen System (Dako Omnis). Samples were counterstained with hematoxylin, incubated in ethanol and xylene solution of ascending concentration, and finally fixed.
4
Detailed Immunohistochemical Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All clinical tissues or xenografts were fixed at 4% PFA Fix Solution for 2 h, then sliced into 5-mm sections after being embedded with paraffin. The paraffin sections were deparaffinized in xylene and rehydrated in a graded alcohol series, boiled with 10mmol -1 of citrate buffer (pH 8.0) for 20min and treated with Endogenous Peroxidase Blocking Buffer (Beyotime) for 10min. The steps were performed using the Envision two-step method using the GTvision TM III detection system and DAB Color kit (Gene Tech, Shanghai, China) or Multiplex IHC Detection (TSA amplification Kit) (Abcam). Antibody of interest was used; PBS was used as the negative control.
Images were captured using an Olympus BX51 microscope (Olympus, Japan), and immunoreactivity was evaluated with IHC Profiler as an Image J plug-in in a blinded manner. The evaluation was based on staining intensity and the extent of staining. The staining intensity of colored cells was assessed as follows: 0 (colorless/negative), 1 (light yellow/low), 2 (dark yellow/moderate), and 3 (yellowish-brown/high).
Images were captured using an Olympus BX51 microscope (Olympus, Japan), and immunoreactivity was evaluated with IHC Profiler as an Image J plug-in in a blinded manner. The evaluation was based on staining intensity and the extent of staining. The staining intensity of colored cells was assessed as follows: 0 (colorless/negative), 1 (light yellow/low), 2 (dark yellow/moderate), and 3 (yellowish-brown/high).
5
Immunohistochemistry of Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thin sections of tissue samples were prepared for immunohistochemistry as previously described [63 (link)]. Briefly, tissue sections were dewaxed for 20 min in xylene and rehydrated using alcohol. Sections were then incubated with endogenous peroxidase blocking buffer (Beyotime) and treated for antigen retrieval for 20 min at 98 °C. Sections were treated with the primary antibodies used for western blotting overnight at 4 °C, then incubated with corresponding secondary antibodies for 30 min at room temperature. Representative images were chosen based on analysis from more than five different fields of view.
6
Immunohistochemical Analysis of Tumor CD3 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were euthanized, and the spleen and tumor tissues of the mice were removed and soaked overnight in 4% formalin. The tissues were fixed with ethanol and xylene at a gradient concentration and then embedded in paraffin wax. A 4–5 mm tissue section was laid flat on adhesive glass slides. Tumor sections were treated with endogenous peroxidase blocking buffer (Beyotime, China) for 10 min to reduce endogenous peroxidase activity. Slides were incubated overnight with rabbit anti-CD3 primary antibody (1:400, Cell Signalling Technology, USA) at 4 °C. Goat anti-rabbit secondary antibody was incubated for 40 min at room temperature. Finally, the sections were visualized with diaminobenzidine (DAB) and haematoxylin staining successively. The state of the section could be observed and imaged under a microscope.
7
Immunohistochemical Analysis of Pulmonary Fibrosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice or IPF paraffin-embedded tissue sections were deparaffinized and then permeabilized with 0.3% Triton X-100 (5 min), followed by blocking with endogenous peroxidase blocking buffer (10 min) (Beyotime), antigen retrieval was carried out by heat mediation in a citrate buffer solution (pH = 6) (20 min). Sections were closed in a blocking buffer (60 min) (Beyotime) and immunostained with anti-α-SMA (1:100; Abcam, ab240654), anti-MMP19 (1:100; Affinity, AF0215), anti-ET1 (1:100; Abcam, ab2786), anti-SDF1 (1:100; Affinity, AF5279), anti-collagen I (1:100; Abcam, ab138492), anti-collagen IV (1:100; Abcam, ab6586), anti-CXCR4 (1:100; Abcam, ab181020), anti-EMR1 (1:100; Affinity, DF2789) overnight at 4 °C, followed by incubation for 1 h at RT with biotin-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies followed by incubating with SABC (1:100; Beyotime), and then visualized by DAB stain (Beyotime). Cell nuclei were labeled with hematoxylin.
8
Immunohistochemical Analysis of TNF-α and Ki-67
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemistry, we used antibodies to TNF-α (1:200, Santa Cruz, Texas, USA) and Ki-67 (1:200, Abcam, Cambridge, UK). Para n section was rehydrated and endogenous peroxidase activity was blocked for 30 minutes by endogenous peroxidase blocking buffer (Beyotime Biotechnology, Shanghai, China). Primary antibody was incubated at 4 °C overnight, followed by 30 minutes for biotinylated secondary antibody (1:500, Abcam, Cambridge, UK). All specimens were counterstained with hematoxylin-eosin (HE) staining solution (Beyotime Biotechnology), then neutral gum sealed piece for storage. Images were obtained after scanning the section through Pannoramic MIDI (3D-Histech, Budapest, Hungary) and analyzed with ImageJ software.
9
Immunohistochemical Analysis of Sertoli Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After baking, dewaxing, and ethanol treatment, the slides were immersed in Citrate Antigen Retrieval Solution (Beyotime, Shanghai, China), and then bathed in boiling water for 30 min. The slides were blocked in Endogenous Peroxidase Blocking Buffer (Beyotime, Shanghai, China) for 10 min, treated with 0.3% Triton-X for 10 min, and then blocked in goat serum (Gibco, Carlsbad, CA, USA) at 37°C for 30 min. After that, the slides were incubated overnight with primary antibodies [GATA4 (1:200) or Ki67 (1:100)] (Abcam, Cambridge, MA, USA) at 4°C and then with secondary antibody for 30 min at 37°C. After treatment with freshly prepared diaminobenzidine working solution (ZSGB-BIO, Beijing, China) and redyeing with hematoxylin, the dyed slides were dehydrated with ethanol and transparent with xylene, followed by mounted with neutral gum. The slides were observed and photographed by microscope. Sertoli cells in 20 spermatogenic tubules were counted for each slide.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!