As previously mentioned, immunohistochemical (IHC) staining was carried out. To summarize, 4 μm thick slices of paraffin-embedded tumor samples from the mouse model experiment and clinical ESCC tissues were cut. After deparaffinization, the sections were rehydrated. Following antigen retrieval, the slices were incubated overnight at 4 °C with anti-Nur77 (1:200, Cat# NB100-56745, Novus), anti-IRF1 (1:100, Cat# 11335-1-AP, Proteintech), anti-PD-L1 (1:5000, Cat# 66248-1-lg, Proteintech), anti-Ki67 (1:300, Cat# GB121141, Servicebio) anti-PCNA (1:1000, Cat# GB12010, Servicebio) and anti-CD8 (1:400, Cat# GB15068, Servicebio) antibodies. Following multiple washes, the sections were incubated for 1 h at room temperature with an HRP-conjugated secondary antibody and stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Servicebio, Wuhan, China). The slides were imaged using a microscope (Olympus BX43F, Japan), and the images were processed using CaseViewer software.
Anti irf1
Manufactured by Proteintech
Sourced in United States
Anti-IRF1 is a primary antibody that recognizes the interferon regulatory factor 1 (IRF1) protein. IRF1 is a transcription factor that plays a role in the regulation of genes involved in immune and inflammatory responses. The Anti-IRF1 antibody can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and immunofluorescence, to detect and study the expression and localization of the IRF1 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti pcna, by Wuhan Servicebio Technology (1 mentions)
Bx43f, by Olympus (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Wuhan Servicebio Technology (1 mentions)
Anti ki67, by Wuhan Servicebio Technology (1 mentions)
Anti cd8, by Wuhan Servicebio Technology (1 mentions)
Anti nur77, by Novus Biologicals (1 mentions)
Anti pd l1, by Proteintech (1 mentions)
Anti jak1, by Abcam (1 mentions)
Anti irf3, by ABclonal (1 mentions)
Rabbit anti flag antibody, by Proteintech (1 mentions)
Anti phospho irf3, by Cell Signaling Technology (1 mentions)
Anti ifit3, by Proteintech (1 mentions)
Anti phospho jak2, by Abcam (1 mentions)
Cell counting kit cck 8, by Biosharp (1 mentions)
Anti ifitm1, by Proteintech (1 mentions)
Anti jak2, by Abcam (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Anti actin, by R&D Systems (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
4 protocols using anti irf1
1
Immunohistochemical Staining of Tumor Samples
2
Interferon Signaling Pathway Analysis
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Human IFN-β recombinant protein (rhIFN-β) and human IFN-λ1 recombinant protein (rhIFN-λ1) were purchased from Abbkine (Wuhan, China) and MedChemExpress (Shanghai, China). The Cell Counting Kit (CCK-8) was obtained from Biosharp (Anhui, China). Pyridone 6 (JAK inhibitor I) (A13457) was purchased from Adooq Biosciences (Irvine, CA, USA). Antibodies used for Western blotting, anti-phospho-STAT1 (S727), anti-STAT1, and anti-IFI35 antibodies (all rabbit polyclonal antibodies), were obtained from Abmart (Shanghai, China). Anti-IFIT2, anti-TLR3, anti-IRF7, anti-IRF3, anti-USP18, and anti-ISG15 antibodies (all rabbit) were purchased from ABclonal (Wuhan, Hubei, China). Anti-JAK1, anti-JAK2, anti-phospho-JAK1, and anti-phospho-JAK2 antibodies (all rabbit) were purchased from Abcam (Cambridge, MA, USA). Anti-MDA5 (IFIH1) and anti phospho-IRF3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-RIG-I (DDX58), anti-IFIT1, anti-IFIT3, anti-IFIT5, anti-IRF1, anti-IFITM1, and anti-β-actin antibodies were purchased from Proteintech (Chicago, IL, USA), and the antibodies used are listed in Additional file 7 : Table S7. The monoclonal antibody against the JEV envelope (E) was kindly provided by Shengbo Cao (Huazhong Agricultural University, Wuhan, China). For immunofluorescence, rabbit anti-FLAG antibody was purchased from Proteintech.
3
Western Blot Analysis of IRF1 Regulation
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Cells were trypsinized, pelleted and washed twice with cold phosphate-buffered saline (PBS). Cell pellets were then lysed in cold low ionic strength buffer (50 mM NaCl, 1% IGEPAL, 10% Glycerol, 10 mM HEPES, pH7.4) with fresh added proteinase inhibitor cocktail (1:100 vol/vol dilution, Sigma P8340), 1 mM DTT and 1 mM PMSF (Sigma P7626). After determination of protein concentration, samples were denatured in SDS sample buffer, heated at 95°C for 5 min, and fractionated using Criterion TGX 4-15% PAGE gels. Proteins were transferred to PVDF membranes and probed with anti-IRF1 (Proteintech 11335-1-AP, 1:500), anti-FLAG M2 (Sigma F1804, 1:1000) or anti-actin (937215, R&D, 1:5000) antibodies (Ab). Anti-TBP Ab (Biolegend 668306, 1:1000) was used as a loading control. Blots were imaged and bands quantified.
4
Western Blot Analysis of Immune Checkpoints
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Tissues or cells were lysed in RIPA buffer (catalog no. KGP702; Keygentec) containing protease and phosphatase inhibitor cocktail (catalog no. P1045; Beyotime) and clarified by centrifugation (12,000×g for 30 min at 4 °C). Protein concentrations were measured using a Pierce BCA Protein Assay Kit (catalog no. KGP902, Keygentec). Protein lysates (30 μg) were subjected to western blotting and protein bands were visualized using a Tianneng imaging system. The following primary antibodies were used: anti-VTCN1 (B7H4) (Proteintech, catalog no. 12080-1-AP), anti-PD-L1/CD274 (Proteintech, catalog no. 66248-1-Ig), anti-IRF1 (Proteintech, catalog no. 11335-1-AP), anti-IFNGR1 (Proteintech, catalog no. 10808-1-AP), anti-IFNGR2 (Proteintech, catalog no. 10266-1-AP), anti-JAK2 (Proteintech, catalog no. 17670-1-AP), anti-STAT1 (Proteintech, catalog no. 10144-2-AP), anti-CD86 (Proteintech, catalog no. 26903-1-AP) and anti-GAPDH (Proteintech, catalog no. 60004-1-Ig). The secondary antibody used was goat anti-mouse IgG (H + L)-HRP (Ray Antibody Biotech, catalog no. RM3001) and goat anti-rabbit IgG (H + L)-HRP (Ray Antibody Biotech, catalog no. RM3002). Because multiple cell lines and target proteins need to be detected, and some proteins have similar molecular weights, the blots have to be cut off before hybridization with antibodies, and Western blotting has to be carried out several times.
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