NSCLC tissues from patients were fixed using 10% formaldehyde for 30 at 37°C, washed with PBS (0.01 mmol/l, pH 7.4) and followed with embedding in paraffin wax. Tissues were deparaffinized in xylene and rehydrated in grade alcohols. Tissues were cut into 4-µm thick sections and antigen retrieval was performed using Antigen Retrieval Reagents (cat. no. CTS015; Bio-Rad Laboratories, Inc.). The sections were washed with PBS for 10–15 min at 37°C and subsequently blocked using 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 2 h at 37°C. Tumor sections were incubated with CD4 (1:1,000 dilutions, Clone 4B12; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and CD8 (1:1,000 dilutions, Clone C8/144B; Dako; Agilent Technologies, Inc.), P53 (1:500 dilutions; ab32049), VEGFR (1:500 dilutions; ab36844), FGFR (1:500 dilutions; ab10646), PDGFR-β (1:500 dilutions, ab220745; all from Abcam) for 12 h at 4°C. The sections were washed three times with PBS for 3 min at room temperature and were incubated with HRP-labeled secondary goat anti-rabbit antibodies (1:2,000, ab150077; Abcam). Sections were visualized using ZEISS LSM 510 confocal microscope at ×40 magnification.
Ab36844
Manufactured by Abcam
Sourced in United States
Ab36844 is a recombinant monoclonal antibody targeting Histone H3 (trimethyl K27). The antibody is produced in HEK293 cells and is purified by protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Clone c8 144b, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab10646, by Abcam (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Ab32049, by Abcam (1 mentions)
Ab220745, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab37150, by Abcam (1 mentions)
Ab39256, by Abcam (1 mentions)
Ab85051, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab73734, by Abcam (1 mentions)
Bca kit, by Yeasen (1 mentions)
Anti ki67, by Cell Signaling Technology (1 mentions)
3 protocols using ab36844
1
Immunohistochemical Analysis of NSCLC Tissue Samples
2
Quantitative Protein Expression Analysis
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Total protein was extracted from both the tissues and cells in strict accordance with the instructions of the efficient radio‐immunoprecipitation assay (RIPA) lysis buffer (R0010, Beijing Solarbio Life Sciences Co Ltd). Next, protein concentration was determined in each sample using a bicinchoninic acid (BCA) kit (20201ES76, Yeasen Biotechnology Co Ltd). Protein transfer onto a polyvinylidene fluoride (PVDF) membrane was performed following isolation by polyacrylamide gel electrophoresis (PAGE) and subsequently sealed with 5% BSA at room temperature for 1 hours. Incubation was subsequently conducted on the membrane with diluted primary rabbit anti‐human antibodies (Abcam Inc) to BTG2 (ab85051, 1:800), vascular endothelial growth factor (VEGF, ab39256, 1:1000), VEGF receptor (VEGFR, ab36844, 1:1000), matrix metalloproteinase‐2 (MMP‐2, ab37150, 1:1000) and MMP‐9 (ab73734, 1:1000) overnight at 4℃. The membrane was then further incubated with HRP‐labelled goat anti‐rabbit IgG diluent (ab205718, 1:20 000, Abcam Inc) at room temperature for 1 hours. ImageJ 1.48 u software (National Institutes of Health) was applied for protein quantitative analysis.
3
Immunohistochemical Analysis of Ki67 and VEGF
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The paraffin-embedded tissue was cut into 5 μm-thickness sections. IHC staining was performed according to the previous method. Briefly, sections were dewaxed with xylene and hydrated with gradient ethanol. The sections were then repaired with trypsin and incubated with primary antibodies anti-Ki67 (#9027, 1:400, Cell Signaling Technology, USA) and anti-VEGF (ab36844, 1:500, Abcam, Cambridge, UK) at 4°C overnight. The sections were then probed with a secondary antibody. After staining, the sections were dehydrated, clarified with xylene and fixed with resin.
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