Brain specimens were fixed in 4% paraformaldehyde for 48h at 4°C and then dehydrated in PBS supplemented with 30% sucrose for 48h at 4°C. The tissue sections were embedded in paraffin wax and mounted on slides, and the slides were heated to 60°C, deparaffinized, and rehydrated in xylene and graded ethanol, heated for antigen retrieval, and treated with an endogenous peroxidase blocking solution. The sections were then incubated with primary anti-RABV P antibody (laboratory prepared, 1:500) overnight at 4°C. Next, the sections were incubated with HRP-conjugated antirabbit secondary antibodies (BioPM, PMK-013-090, 1:200) for 30min, followed by two washes in PBS. After washing, the sections were incubated with diaminobenzidine (Servicebio, G1211) for color development and then photographed and analyzed using an XSP-C204 microscope (CIC).
Diaminobenzidine
Manufactured by Wuhan Servicebio Technology
Sourced in China
Diaminobenzidine is a chromogen used in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) to produce a brown color reaction. It is a sensitive substrate for the detection of peroxidase enzyme activity.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Wuhan Servicebio Technology (3 mentions)
Biotinylated secondary antibody, by Boster Bio (1 mentions)
Avidin biotin peroxidase, by Boster Bio (1 mentions)
Ab131215, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Biotinylated secondary antibody, by Wuhan Servicebio Technology (1 mentions)
Ab180125, by Abcam (1 mentions)
Ab134958, by Abcam (1 mentions)
Imagej 6, by Media Cybernetics (1 mentions)
Neutral resin, by Wuhan Servicebio Technology (1 mentions)
3 3 diaminobenzidine (dab), by Wuhan Servicebio Technology (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Avidin biotin complex vectastain kit, by ZSGB-BIO (1 mentions)
G1203, by Wuhan Servicebio Technology (1 mentions)
Ds u3, by Nikon (1 mentions)
G5001, by Wuhan Servicebio Technology (1 mentions)
Optical microscope, by Nikon (1 mentions)
11 protocols using diaminobenzidine
1
Immunohistochemical detection of RABV P
2
Immunohistochemical Detection of FHL2 in Ovarian Tissues
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A previously described peroxidase-based immunohistochemistry protocol was used to detect FHL2 expression in paraffin-embedded ovarian tissues [36 (link)]. Anti-FHL2 antibody (Proteintech, Wuhan, China) was used at 1:200. Subsequently, the sections were incubated with biotinylated secondary antibody (1:2000) (Boster Biotechnology, Wuhan, China) and avidin-biotin-peroxidase (Boster Biotechnology, Wuhan, China) before being exposed to diaminobenzidine (Servicebio, Wuhan, China, Wuhan, China) and counterstained with hematoxylin (Servicebio, Wuhan, China).
3
Immunohistochemical Analysis of ACAT2 Expression
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Paraffin-embedded tissues were serially sectioned at 4 μm thickness. They were baked at 65℃ for 2 h, deparaffinized in xylene, and rehydrated through graded alcohol. Subsequently, slices were placed in 3% hydrogen peroxide, heated in 1x sodium citrate antigen retrieval buffer (pH 6.0) by pressure cooker and blocked with 5% bovine serum albumin (Solarbio). The slices were incubated with rabbit anti-ACAT2 monoclonal antibody overnight at 4℃ (1:300; ab131215, Abcam) and then with the MaxVision-HRP rabbit/mouse antibody (Maixin) at room temperature for 1 h. Diaminobenzidine (Servicebio) was used as the final chromogen. Hematoxylin (Servicebio) was applied to counterstain.
All slices were evaluated by two experienced pathologists in a blinded manner. For the assessment of ACAT2, five high-power fields in each specimen were selected randomly, and cytoplasm staining was examined. Immune score equaled to the percentage of positive cells (0, ≤ 5%; 1, 6 − 25%; 2, 26 − 50%; 3, 51 − 75%; 4, 76 − 100%) multiplied by the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong). Immune scores of 0–4 are defined as low expression and 5–12 as high expression [31 (link)].
All slices were evaluated by two experienced pathologists in a blinded manner. For the assessment of ACAT2, five high-power fields in each specimen were selected randomly, and cytoplasm staining was examined. Immune score equaled to the percentage of positive cells (0, ≤ 5%; 1, 6 − 25%; 2, 26 − 50%; 3, 51 − 75%; 4, 76 − 100%) multiplied by the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong). Immune scores of 0–4 are defined as low expression and 5–12 as high expression [31 (link)].
4
Immunohistochemical Profiling of Ribosomal Proteins in Rat and Clinical Samples
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Tissue specimens from rat hind limbs and clinical samples were fixed in 4% paraformaldehyde for 48 h and washed with phosphate buffered saline, then dehydrated in a graded ethanol series, vitrified with dimethylbenzene, and inserted in paraffin. Paraffin sections (4 μm) were deparaffinized in xylene, hydrated with gradient ethanol, and stained with standard H&E, SOFG, or Masson staining procedures. To perform the immunohistochemical staining, tissue slides were deparaffinized and rehydrated followed by antigen retrieval, endogenous peroxidase blocking and serum sealing. Then, the slides were incubated with antibodies against UBA1(1:100, ab180125, Abcam Inc, Cambridge, UK), EIF3E (1:50, ab134958, Abcam Inc, Cambridge, UK), RPL27 (1:50, 14980-1-AP, Proteintech, Chicago, USA), RPL17 (1:300, 67223-1-Ig, Proteintech, Chicago, USA), RPS28(1:50, 14796-1-AP, Proteintech, Chicago, USA) at 4 °C overnight. The next day, all sections were taken out and washed with PBS several times. Then the biotinylated secondary antibody (Servicebio, Wuhan, China) was used for 1 h at room temperature followed by the reaction with diaminobenzidine (Servicebio, Wuhan, China) and hematoxylin to develop color. Histological scores were calculated from the results of staining using Image J 6.0 (Media Cybernetics Corporation, USA) software.
5
Immunohistochemical Analysis of KIRC Tissues
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KIRC tissues were embedded in paraffin to make paraffin sections with a thickness of 4 μm. The sections were stained by the immunohistochemical streptavidin-peroxidase method, and the operation was performed strictly by the kit instructions (Servicebio, China). DAB (Diaminobenzidine, Servicebio, China) chromogenic solution was used for visualization color reaction and counterstained with hematoxylin (Servicebio, China). The sections were dehydrated in different gradients of ethanol. Then neutral resin (Servicebio, China) was used to mount the sections. The sections were observed under an inverted microscope (Olympus, Japan).
6
Immunohistochemical Analysis of Breast Cancer Markers
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Immunohistochemical analysis was performed as reported previously [26 (link)]. Briefly, formalin-fixed paraffin-embedded tissue sections were deparaffinized and rehydrated. After heat-induced antigen retrieval (TE buffer pH 9.0, G1203, ServiceBio, China), the slides were analyzed using an Avidin–Biotin Complex Vectastain Kit (SP9001, ZSGB-Bio, China) as per the manufacturer’s instructions. Primary antibodies, including AR (1:100, 22089-1-AP, Proteintech, China), ER (1:250, 21244-1-AP, Proteintech), and PR (1:100, 25871-1-AP, Proteintech) were used according to the manufacturer’s guidelines. After incubation with a peroxidase-conjugated secondary antibody (Rabbit, 1:100, ServiceBio), the slides were detected using diaminobenzidine (G1212-200T, ServiceBio). Finally, the samples were counterstained with Hematoxylin. The percentage of positive tumor cells was determined in accordance with the pathologic report of Tongji Hospital. All slides were examined by two investigators, who were blinded to all clinicopathologic variables.
7
Immunohistochemical Analysis of Proteins
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The four-μm thick paraffin sections were dewaxed, hydrated, and antigen retrieved. Sections were then blocked with 5% bovine serum albumin (BSA), followed by overnight incubation of primary antibody at 4 °C. After washing, the sections were incubated with secondary antibodies, reacted with diaminobenzidine (Servicebio) and stained with hematoxylin to visualize the nuclei. Sections were observed by a light microscope. Proteins were quantified using Image-Pro plus 6.0.
8
Immunohistochemical Analysis of Tumor Immune Markers
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The protocol was adjusted based on our previous study [32 (link)]; 5-µm thick paraffin-embedded sections of the tumor were deparaffinized and rehydrated with paraffin, blocked with 3% H2O2 for 10 min, sealed with 3% BSA (G5001; Servicebio, Wuhan, China) for 30 min, and were then incubated overnight at 4 °C with primary antibodies against CD3e (#GB13014), CD4 (#GB13064-2), CD8 (#GB13429), CD20 (#GB11540), and IL-2 (#GB11114). The sections were incubated with goat anti-rabbit IgG(H+L) (peroxidase/HRP-conjugated; #G1213). After staining with diaminobenzidine (#G1212; Servicebio), the slices were re-stained with hematoxylin for 3 min, dehydrated, and blocked. The slides were examined using a microscope (Nikon DS-U3, Nikon). All of the antibodies were obtained from Servicebio.
9
Immunohistochemical Analysis of Hypothalamic Proteins
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The immunohistochemistry protocol was conducted as described previously (Eraky et al., 2021 (link)). Briefly, paraffin sections (5 μM thick) of the hypothalamus were deparaffinized, rehydrated, and then immunostained with rabbit Irs-1 (Servicebio, Wuhan, China) polyclonal and Srsf3 monoclonal antibodies (Thermo Fisher Scientific, Runcorn, Cheshire, United Kingdom). After three washes in phosphate-buffered saline (pH 7.4), slides were subsequently incubated with corresponding species of horseradish peroxidase-labeled secondary antibody. Diaminobenzidine (Servicebio, Wuhan, China) was incubated as a chromogenic reagent. The staining time was controlled under the microscope. Positive staining was brownish yellow. Hematoxylin was used for counterstaining and visualized by a Nikon optical microscope (Nikon, Tokyo, Japan). Average optical density (AOD) value was used to semi-quantify the protein expression on immunohistochemical staining by ImageJ software (version 1.45) (NIH, Bethesda, MD, United States) (Liu et al., 2015 ).
10
Immunohistochemical Staining of Ly-6G
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IHC was performed by treating the paraffin-embedded tissue sections with 3% H2O2 for 15 min and then subjecting them to microwave Ag retrieval. The slides were blocked with 3% BSA for 30 min and incubated with rabbit anti-mouse anti Ly-6G Ab overnight at 4 °C. After three washes with PBS, the samples were first incubated with HRP-conjugated goat anti-rabbit antibody and then with diaminobenzidine (Servicebio, Wuhan, China) as a substrate and counterstained with haematoxylin. Images were captured using an Olympus microscope (Olympus, Tokyo, Japan). Five fields per slide were selected randomly from a single mouse and then evaluated by an experienced pathologist blinded to the assignment of mice to treatments.
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