Human brain tissues were fixed in 4% paraformaldehyde at room temperature for 48 h and embedded in paraffin and cut into sections. The thickness of sections used for immunohistochemical staining was 6-µm and these sections were cut using a paraffin section machine (cat. no. HM340E; Thermo Fisher Scientific, Inc.). After paraffin sections were deparaffinized at 65°C for 2 h, hydrated with 100/95/75% ethanol for 10 min each, antigen recovery was performed by exposing sections to 0.01 mol/l citrate buffer (pH 6.0) at 100°C for 20 min. Endogenous peroxidase was removed by treatment with 3% H2O2. Samples were blocked with 1% BSA (Amresco, LLC) at room temperature for 1 h and were then incubated with primary antibodies at 37°C overnight. Following 3 washes with PBS, the sections were incubated with secondary antibody for 1 h at room temperature. DAB (Wuhan Servicebio Technology, Co., Ltd.) was used for dyeing and the nuclei were stained by hematoxylin at room temperature for 30 sec. Images were captured with an Olympus BX51 fluorescence microscope (Olympus Corporation).
3 3 diaminobenzidine (dab)
Manufactured by Wuhan Servicebio Technology
Sourced in China, United States
The DAB (3,3′-Diaminobenzidine) is a chromogenic substrate used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) techniques. It produces a brown-colored precipitate upon oxidation, allowing for the visualization and localization of the target antigen or enzyme in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Wuhan Servicebio Technology (9 mentions)
Bx51 microscope, by Olympus (4 mentions)
Anti dig hrp igg fraction monoclonal, by Jackson ImmunoResearch (3 mentions)
Bovine serum albumin (bsa), by Wuhan Servicebio Technology (3 mentions)
Neutral resin, by Wuhan Servicebio Technology (2 mentions)
Ab15580, by Abcam (2 mentions)
Gb23303, by Wuhan Servicebio Technology (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Hm340e, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
C14190500bt, by Thermo Fisher Scientific (1 mentions)
Vcam 1, by Wuhan Servicebio Technology (1 mentions)
Mmp 9, by Wuhan Servicebio Technology (1 mentions)
Icam 1, by Wuhan Servicebio Technology (1 mentions)
Mmp 2, by Wuhan Servicebio Technology (1 mentions)
Optical microscope, by Nikon (1 mentions)
Hrp conjugated goat anti rabbit igg, by Abcam (1 mentions)
Horseradish peroxidase, by Wuhan Servicebio Technology (1 mentions)
67 protocols using 3 3 diaminobenzidine (dab)
1
Immunohistochemical Analysis of Human Brain Tissue
2
Immunohistochemical Analysis of Tissue Sections
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The paraffin-embedded sections were de-waxed, soaked in recovery buffer containing EDTA antigen (Wuhan Servicebio Technology Co., Ltd.), and incubated with the primary antibodies at 4°C overnight. The sections were then incubated in HRP-secondary antibody (1:200; cat. no. G1213, Wuhan Servicebio Technology Co., Ltd.) at room temperature for 2 h. Following incubation with DAB (cat. no. G1212, Wuhan Servicebio Technology Co., Ltd.) and hematoxylin (cat. no. G1004, Wuhan Servicebio Technology Co., Ltd.), the sections were sealed with neutral resin (cat. no. WG10004160, Wuhan Servicebio Technology Co., Ltd.). Images was obtained using a light microscope (Leica Microsystems GmbH). The primary antibodies used were the following: MPO (1:200, cat. no. YT5351, ImmunoWay), CD68 (1:200, cat. no. ab283654, Abcam), caspase-3 (1:200, cat. no. A0214, ABclonal), poly(ADP-ribose) polymerase 1 (PARP1; 1:200, cat. no. A19596, ABclonal) and TGF-β (1:200, cat. no. bs-0103R, BIOSS).
3
Immunohistochemical Analysis of Cervical Cancer
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Human cervical cancer tissue sections were retrospectively obtained from surgical resections that were fixed in buffered formalin, embedded in paraffin, and stored at the Zhujiang Hospital, Southern Medical University, Guangzhou, China. The corresponding clinical data were obtained from medical records and identified. The ethics committee of Zhujiang Hospital approved the use of the clinical specimens. For IHC staining, antigen retrieval was performed by heat treatment in a microwave oven for 21 min in Tris-ethylene diamine tetraacetic acid (EDTA) buffer solution (0.05 mol/L Tris, 0.001 mol/L EDTA; pH 8.5). Endogenous peroxidase activity was inactivated using 0.3% H2O2 for 10 min followed by washing with PBS (Gibco, C14190500BT). After blocking by 5% BSA for 20 min, the slides were incubated overnight at 4°C with the following primary antibodies used (proteintech, 22092-1-AP-50UL). After washing with PBS, the sections were incubated with HRP conjugated goat anti-rabbit IgG secondary antibodies (Cell Signalling, 7074) for 50 min. Finally, immunoreactivity was detected using 3,3-diaminobenzidine (Servicebio, G1211), followed by re-staining with hematoxylin. Images were obtained by using 3D HISTECH (Pannoramic MIDI II).
4
Immunohistochemical Analysis of CD8+ T Cells in Tumor Tissues
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The resected tumor tissues were fixed in 4% PFA (Cat# G1101, Servicebio) and embedded in paraffin blocks. Sections were cut at a thickness of 5 µm from the paraffin-embedded blocks, deparaffinized by immersion in xylene (10 min, ×2), and rehydrated in graded ethanol (100% for 3 min, ×2; 95% for 3 min; 70% for 3 min; 50% for 3 min) and incubated with 3% (w/v) hydrogen peroxide to block endogenous peroxidase activity. After that, the sections were incubated with a rabbit antimouse CD8α antibody (1:200 dilution; Cat# ab217344, Abcam, USA) and an HRP goat antirabbit IgG secondary antibody (1:200 dilution; Cat# G1213, Servicebio). Finally, the sections were stained with 3′,3-diaminobenzidine (Cat# G1212-2, Servicebio) and counterstained with 37% (w/v) hematoxylin (Cat# G1004, Servicebio).
5
Immunohistochemical Analysis of FOXO1 and KI67
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Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, then sliced into 4.0-μm sections. Subsequently, sections were deparaffinized, rehydrated, and incubated with primary antibodies over night at 4°C. Following incubation, the sections were washed three times with PBS and incubated with secondary antibodies. Finally, all sections were stained with 3,3-diaminobenzidine (Servicebio, China) and visualized using a light microscope (Olympus, Japan). The primary antibodies used for immunohistochemistry were anti-FOXO1 (2880, CST, USA) and anti-KI67 (ab15580, Abcam). The average integrated optical density (IOD) was determined for quantitation using Image-Pro Plus software (Version 6.0). The relative mean IOD of each group was divided by that of the control group.
6
Immunofluorescence and Immunohistochemistry Analysis
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KGN cells were fixed with 4% paraformaldehyde and then treated with 10% goat serum for 30 min to allow for cell immunofluorescence. Then, they were treated with anti-RPL11 and anti-MDM2 antibodies (1:50) at 4 ℃. On the second day, the cells were rinsed with PBST and then incubated with secondary an Cy3 conjugated Goat Anti-Rabbit IgG (H + L) and FITC conjugated Goat Anti-Mouse IgG (H + L) for 1 h. The nuclei were stained with 4',6-diamidino-2-phenylindole for 5 min. Next, the cells were visualized using a confocal microscope.
To stop endogenous peroxidase activity, the paraffin sections were boiled in an antigen retrieval solution in a microwave oven, followed by treatment with 0.3% hydrogen peroxide for 10 min. After 30 min, the sections were incubated with 10% normal goat serum and treated with anti-BOP1 (1:50), anti-LC3 (1:100), and anti-SQSTM1 (1:100) antibodies overnight. The paraffin sections were then washed thrice with PBST, followed by treatment with secondary antibodies for 2 h. The sections were then stained with hematoxylin and submerged in 3,3-diaminobenzidine (Servicebio).
To stop endogenous peroxidase activity, the paraffin sections were boiled in an antigen retrieval solution in a microwave oven, followed by treatment with 0.3% hydrogen peroxide for 10 min. After 30 min, the sections were incubated with 10% normal goat serum and treated with anti-BOP1 (1:50), anti-LC3 (1:100), and anti-SQSTM1 (1:100) antibodies overnight. The paraffin sections were then washed thrice with PBST, followed by treatment with secondary antibodies for 2 h. The sections were then stained with hematoxylin and submerged in 3,3-diaminobenzidine (Servicebio).
7
Immunohistochemical Analysis of Cardiac Markers
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Samples from the left ventriculum were fixed in 4% paraformaldehyde at 25°C, embedded in paraffin, and cut into 3 μm thick sections. After deparaffinization in xylene, the tissues were subjected to antigen retrieval using 10 mM citrate-phosphate buffer (pH 6.0) and incubated in 3% H2O2 for 25 min. Then, the sections were blocked with 1% bovine serum albumin in PBS for 10 min. Following incubation with a prediluted biotinylated pan-specific universal secondary antibody (Servicebio, China) for 50 min at room temperature, sections were incubated overnight with mouse monoclonal ICAM-1 (1 : 800, Servicebio), VCAM-1 (1 : 400, Servicebio), MMP-2 (1 : 2000, Servicebio), and MMP-9 (1 : 800, Servicebio) antibody at 4°C. The tissues were washed with PBS, incubated in the streptavidin/peroxidase complex for 5 min and then washed with PBS again, before being washed with 3, 3′-diaminobenzidine (Servicebio, China) for 7 min. Sections were stained with hematoxylin for 3 min, then washed, and mounted. Images were captured using a fluorescent microscope (XSP-C204, CIC, Germany).
8
Histological Analysis of Pancreatic AMPK and mTOR
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At the end of the experiment, the pancreas of each mouse was quickly isolated after blood collection. The pancreas from mice in each group was obtained and fixed in 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin, and sliced into 4 µm sections. The sections were prepared and used to perform hematoxylin-eosin (H&E), Masson, and Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling (TUNEL) staining according to the manufacturer’s instructions. For immunohistochemical analysis, paraffin sections were routinely dewaxed and hydrated, antigen was repaired with citrate buffer, and endogenous peroxidase was closed with 3% H2O2. Next, the sections were incubated with Rabbit anti-p-AMPK antibody (Affinity Biosciences, 1:200) or anti-p-mTOR antibody (Affinity Biosciences, 1:200). HRP-conjugated Goat Anti-Rabbit IgG (Abcam, ab97080, 1:5000) was used as secondary antibody for 30 min and visualized by the 3.3′‐diaminobenzidine (Servicebio, G1212-200T). It was observed using an optical microscope (Nikon, Japan). Positive staining of TUNEL and IHC was quantified by Image Pro Plus software.
9
Immunodetection of Anaplasma capra in Cells
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Cell smears were prepared from the uninoculated and inoculated human erythrocytes, HL-60, and TF-1 cells that were collected at different time points. The A. capra-positive serum samples collected from the goat were used as the primary antibody and incubated at 4 °C overnight. Subsequently, HRP-conjugated Rabbit Anti-Goat IgG (H + L) (Servicebio, Wuhan, China) was applied to the smears and incubated at 37 °C for 1 h. The antibody was visualized using 3,3′-diaminobenzidine (Servicebio, Wuhan, China), and the images recorded using a light microscope (Nikon, Tokyo, Japan). As negative controls, the antiserum from the A. capra-negative goat and A. capra-negative blood smears were processed in the same manner and examined.
10
Immunohistochemical Analysis of LGG Samples
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Paraffin-embedded tumor tissues of 20 of 77 LGG samples used for RNA sequencing from the Xiangya in-house dataset were further collected for immunohistochemistry (IHC) staining. Briefly, tissue sections were placed in citric acid antigen repair buffer (pH 6.0) in a microwave oven for antigen retrieval. Slices were placed in a 3% hydrogen peroxide solution to block endogenous peroxidase. 3% bovine serum albumin (BSA) was used as a blocking reagent. The sections were incubated with primary antibodies against CD8 (Mouse, 1:1000, 66868-1-Ig, Proteintech, China), PD-1 (Rabbit, 1:800, 18106-1-AP, Proteintech, China), PD-L1 (Mouse, 1:1000, 66248-1-Ig, Proteintech, China). The sections were then incubated with a horseradish peroxidase-conjugated secondary antibody (1:200, GB23303, Servicebio, China). 3-3'-diaminobenzidine (G1211, Servicebio, China) was finally used for coloration, and hematoxylin was used for counterstaining cell nuclei.
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