Cell proliferation was performed by immunostaining with Ki67 antibody. Briefly, cells cultured on coverslips were fixed in 10% neutral buffered formalin and permeabilised with 0.2% triton X-100. After blocking with serum-free blocking solution (Thermo Fisher Scientific) for 10 min, cells were incubated with primary antibody against Ki67 (0.29 µg/mL, rabbit monoclonal IgG; Abcam) antibody and counterstained with 4′6-diamidino-2-phenylindole (DAPI, 1 µg/mL; Thermo Fisher Scientific) for nuclear staining. Images were taken using BX-61 Olympus fluorescence microscope (Tokyo, Japan). The number of proliferative cells (Ki67 positive) was counted from three random fields and expressed as a percentage over total number of cells (DAPI positive).
Rabbit monoclonal igg
Manufactured by Abcam
Sourced in United States
Rabbit monoclonal IgG is a laboratory reagent used for various research applications. It is a purified immunoglobulin G (IgG) antibody derived from rabbits.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bx61 fluorescence microscope, by Olympus (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Anti ly6g clone 1a8, by BD (1 mentions)
Normal rabbit igg, by Abcam (1 mentions)
Evos fl microscope, by Thermo Fisher Scientific (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Anti cd45 clone 30 f11, by Thermo Fisher Scientific (1 mentions)
C ebpδ, by Abcam (1 mentions)
C ebpβ, by Abcam (1 mentions)
Ghost dye, by Thermo Fisher Scientific (1 mentions)
Anti ly6c clone hk1, by Thermo Fisher Scientific (1 mentions)
Anti cd11b clone m1 70, by BioLegend (1 mentions)
Buffered formalin, by Merck Group (1 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
A5441, by Merck Group (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
5 protocols using rabbit monoclonal igg
1
Ki67 Immunostaining for Cell Proliferation
2
Multiparameter Analysis of Kidney Cells
3
Ki67 Immunohistochemistry in Mouse Tumor Tissue
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Tissue microarray (TMA) blocks were selected tissue cores (diameter 2 mm) from mouse tumor paraffin blocks. To make slides for IHC, all tissue samples were fixed in buffered formalin (Sigma–Aldrich, MO, USA) and embedded in paraffin. Paraffin-embedded tissues were deparaffinized in xylene and rehydrated in graded alcohol (100, 90, 80, and 60%). Antigen retrieval (10 min in boiling water) was performed, and sodium citrate was used as the retrieval buffer. The primary Ki67 antibody used was rabbit monoclonal IgG (Abcam, MA, USA). Immunostaining was performed with a Rabbit IgG/Mouse IgG Vectastain Elite ABC Kit (Vector Laboratories, CA, USA). Vectastain Elite ABC Reagent was added (30 min at RT), and immunoreaction was detected using 3,3’-diaminobenzidine (Sigma–Aldrich, MO, USA) as a chromogen. Then, the TMA slides were counterstained with Mayer’s hematoxylin (Dako, CA, USA).
4
Dual Staining of Muscarinic Receptors and Neurofilaments
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The bladder sections were then double stained with the primary antibodies to muscarinic acetylcholine receptor M2 (1:100, rabbit monoclonal IgG; Epitomics) and Neurofilament (1:200, mouse monoclonal IgG2b; Novus) at 4 °C overnight, then incubated with secondary antibody (1:800; Invitrogen) conjugated to fluorescein isothiocyanate (FITC) for M2, conjugated to rhodamine for Neurofilament. The nuclei of the cells were counterstained with DAPI.
5
Western Blot Analysis of Protein Expression
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Western blot analysis was performed as reported [32 (link)]. Cells were harvested with RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Na-desoxycholate, 0.1% SDS, 1 mM NaF, phosphatase, and protease inhibitor cocktail tablets (Roche, Mannheim, Germany)). The following antibodies were utilized: mouse monoclonal antibody against β-actin (A5441), rabbit polyclonal antibody against human chorionic gonadotropin (β-hCG, SAB4500168) from Sigma-Aldrich (Taufkirchen, Germany), and mouse monoclonal antibody against GAPDH (GTX627408) from GeneTex (Eching, Germany). The RITA antibody was commercially designed and produced (rabbit monoclonal IgG, Epitomics, Burlingame, USA) as stated [11 (link)]. The ImageJ 1.48v software (National Institutes of Health, Bethesda, Maryland, USA) was used for densitometry measurements of Western blot analysis.
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