Fresh target tissues were collected and fix it in 10% neutral buffered formalin for an appropriate period. Transfer the tissue into a labeled cassette and dehydrate it by placing it in a series of increasing concentrations of alcohol for gradual dehydration. Embed the tissue in molten paraffin wax and allow it to solidify. Then, the paraffin-embedded tissue block were cut to 4 μm section and subjected to deparaffinization and rehydration with xylene and graded alcohols. 3% H2O2 was utilized to eliminate endogenous peroxidase after antigen retrieval with five mM citrate buffer. The slides were blocked for 30 minutes at room temperature with goat serum before being treated with primary antibodies overnight at 4 ℃. The slices were rinsed three times in PBS before being treated for two hours at room temperature with a biotinylated secondary antibody. As a chromogen substrate, diaminobenzidine was utilized. Hematoxylin was used to counterstain the sections at the end. Immunohistochemistry was performed using antibodies against IL-17 (ab79056, Abcam), CD163 (ab79056, Abcam), CD64 (ab140779, Abcam), CD1A (17325-1-AP, Proteintech), CD57 (19401-1-AP, Proteintech), CD8 (66868-1-Ig, Proteintech) and CD3 (3F3A1, Proteintech).
Ab79056
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab79056 is a recombinant anti-GAPDH antibody produced in rabbit. It is designed for use in Western blotting, immunohistochemistry, and immunocytochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab6671, by Abcam (5 mentions)
Ab150077, by Abcam (4 mentions)
Ab183685, by Abcam (3 mentions)
Ab20034, by Abcam (3 mentions)
Image pro plus 6, by Media Cybernetics (3 mentions)
Ab115759, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab216327, by Abcam (2 mentions)
Dab chromogen, by Agilent Technologies (2 mentions)
Meyer s hematoxylin, by Agilent Technologies (2 mentions)
Bx51 fl, by Olympus (2 mentions)
Axio imager 2, by Zeiss (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Ab2378, by Abcam (2 mentions)
Ab9811, by Abcam (2 mentions)
Ab22510, by Abcam (2 mentions)
Ab140779, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
K3468, by Agilent Technologies (2 mentions)
Ab9722, by Abcam (2 mentions)
52 protocols using ab79056
1
Immunohistochemical Analysis of Tissue Samples
2
Immunohistochemical Profiling of GBM Tumor
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The GBM tumor tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and subsequently cut into 4 μm sections. The paraffin sections were incubated overnight at 4° C with antibodies according to standard protocols. Two independent, blinded pathologists independently evaluated each section. Two independent blinded pathologists assessed each section separately. Immunohistochemistry was performed using antibodies against PTPN6 (ab32559, Abcam), CD1A (17325-1-AP, Proteintech), IL-17 (ab79056, Abcam), CXCR5 (ab254415, Abcam), CD8 (66868-1-Ig, Proteintech), Tryptase (ab2378, Abcam), CD20 (10252-1-AP, Proteintech), CD45 (60287-1-Ig, Proteintech), FOXP3 (ab20034, Abcam), CD57 (19401-1-AP, Proteintech), CD64 (ab140779, Abcam) and CD163 (ab79056, Abcam).
3
Immunohistochemical Analysis of gH2AX and IL-17
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Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections. Briefly, 4 µm-thick sections were deparaffinized in xylene, rehydrated in graded ethanol, and washed twice with PBS. Endogenous peroxidase activity was blocked by incubating sections with 3% hydrogen peroxide in the dark for 10 min. Slides were incubated overnight at 4 °C with the primary antibody (gH2AX, 1:200, Abcam ab26350, Cambridge, MA, USA; IL-17, 1:400, Abcam ab79056, Cambridge, MA, USA). The sections were washed and incubated at room temperature for 1 h with the secondary antibodies. Finally, the slides were exposed to a substrate chromogen mixture and counterstained with hematoxylin and eosin (H&E). Stained slides were analyzed on an Olympus optical microscope and scored according to the number of positively stained cells.
4
Immunohistochemical Localization of TLR5 and IL-17A
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The colon was fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and cut into 5 mm sections. The sections were baked at 60°C for 12 h and blocked with 10% normal serum/5% BSA, followed by incubation overnight at 4°C in solution with TLR5 (19810-1-AP; Proteintech) and IL-17A (ab79056; Abcam) polyclonal rabbit antimouse antibodies (Proteintech, Wuhan, China), respectively. After washing, the sections were incubated with the goat antirabbit IgG antibody. The sections were then washed again, incubated in the sDAPI working solution at 37°C for 10 min, and were sealed and observed under a laser confocal microscope (ThermoFisher, New York, USA). Three fields (×400) were selected from each section for the positive cell count.
5
Immunofluorescence Staining of Mouse Skin
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Mice skin was freshly excised into small pieces, washed three times in PBS, and embedded in a tissue freezing medium. 10 μm vertical sections were performed on Leica 230 CM1905 cryostat (Leica, Leica Biosystems, Buffalo Grove, IL). Then skin sections were fixed with 4% paraformaldehyde, blocked by 5% BSA and 0.3% Triton X-100 for 1 h, and with anti-IL17 (ab79056; 1:1000; Abcam) as primary antibody incubated overnight at 4°C, goat anti-rabbit IgG H&L (ab150077; 1:1000; Abcam) as secondary antibody. After that, sections were incubated with Hoechst 33342 (5 μg/ml) for 15 minutes and made into the slide for confocal laser scanning microscopy observation (Leica TCS SP8, Solms, Germany).
6
Immunophenotyping of Infiltrating Cells
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Anti-CD3 (Bioss bs-0765R), anti-CD4 (Abcam, ab11815), anti-CD5 (Bioss, bs1113R), anti-CD8 (Abbiotec, P0731), anti-CD11b (Abcam, ab75476), anti-CD45 (Abbiotec, ab08575), anti-TNF-α (Abcam, ab199013), anti-IL-17 (Abcam, ab79056), anti-IL-22 (Abcam, ab106773) and anti-IL-23 (Abcam, ab115759) were used as primary antibodies. Incubation time and dilutions of primary antibodies were optimized according to the manufacturers’ instructions. Positive (+) stained cells were calculated for each animal of the groups and expressed as mm2 with the help of Kameram image analysis software (Kameram 2.1; Argenit, Istanbul, Turkey).
7
Protein Expression Analysis in Mouse Tissues
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Mouse brain tissue and colon tissue were collected from each group. Protein samples were separated on SDS−PAGE gels then electrotransferred to nitrocellulose membranes (Millipore, Burlington, MA, USA). The primary antibodies were hybridized with membranes. The primary antibodies included anti-IL-10 (1:200, ab9969, Abcam, Cambridge, MA, USA) and anti-IL-17 (1:200, ab79056, Abcam, Cambridge, MA, USA). The secondary antibodies incubated with membranes. The ImageJ software were used to scan and analyze protein bands. The bands of brain tissue were normalized to β-tubulin (ab 7291, Abcam, Cambridge, MA, USA), and colon tissue were normalized to β-actin (ab8227, Abcam, Cambridge, MA, USA).
8
Immunohistochemical Analysis of Thrombosis
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IVC tissues containing thrombus were fixed on slides and rinsed with PBS. The slides were blocked in 5% BSA in PBS for 1 h and treated with the primary antibodies at 4°C overnight, namely, rabbit anti-MLKL (p-S345) antibody (1 : 1000; #ab196436; Abcam, Cambridge, United Kingdom), rabbit anti-RIP3 (p-S232) antibody (1 : 50; #ab195117; Abcam, Cambridge, United Kingdom), and rabbit anti-IL-17B antibody (1 : 100; #ab79056; Abcam, Cambridge, United Kingdom). The slides were incubated at 37°C for 40 min with the secondary antibody (goat anti-rabbit IgG antibody, Abcam, Cambridge, United Kingdom) after washing with PBS three times and stained with hematoxylin for 5 min. The images were revealed using HRP-DAB and observed using a Lab.A1 microscope at 200x (Carl Zeiss GmbH, Oberkochen, Germany). The positive expression of the protein was observed by an optical microscope and showed brownish-yellow particles. Five high-power fields were randomly selected for observation using the double-blind method. The Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the positive rate.
9
Immunohistochemical Analysis of Colon Tissue
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Colon tissue sections (4 mm) were deparaffinized in xylene twice for 10 min, rehydrated in a graded ethanol series once for 5 min, incubated in sodium citrate buffer for 10 min for antigen retrieval, blocked with 10% bovine serum albumin for 1 h, and incubated with antibodies against Claudin-1 (1:100; Abcam, Cambridge, UK; ab242370), zonula occludens 1 (ZO-1) (1:100; Abcam; ab276131), Occludin (1:100; Abcam; ab216327), mucin 2 (MUC2) (1:500; Abcam; ab272692), myeloperoxidase (MPO) (1:500; Abcam; ab208670), CD11b (1:100; Abcam; ab133357), IL-17 (2 mg/mL; Abcam; ab79056), IL-10 (10 mg/mL; Abcam; ab189392) overnight, and IL-1β (1:500; Abcam: ab283818). The sections were then incubated with secondary antibodies (SP9000; Zsbio Biotechnology, Beijing, China) for 1 h. The images were acquired using a BX53F microscope (Olympus, Tokyo, Japan).
10
Immunofluorescence Staining of CD4 and IL-17
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For immunofluorescence staining, the sections were incubated with primary antibody: CD4 (RD, BNP2‐25191), IL‐17(Abcam, ab79056) overnight, followed by secondary antibody.
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