In short, after routine dewaxing, antigen retrieval was carried out with 0.1% collagenase from C. perfringens (Sigma, c6885, USA) in a 37°C thermostat for 30 min. The sections were blocked with 5% BSA at room temperature for 20 min. Primary rabbit antibodies were used to visualize Pax7 (Abcam, ab187339, UK; Pax7 : 1% BSA 1:200) + BrdU (Abcam, ab6326, UK; BrdU : 1% BSA 1:200); MyoD (Abcam, ab133627, UK; MyoD : 1% BSA 1:200) + BrdU (Abcam, ab6326, UK; BrdU : 1% BSA 1:200); Pax7 (Abcam, ab187339, UK; Pax7 : 1% BSA 1:200) + Sca1 (Abcam, ab25031, UK; Sca1 : 1% BSA 1:200), overnight at 4°C. Anti‐rabbit IgG [Cell Signaling Technology (CST) 4412, USA, 488; 488 : 5% BSA 1:500 + CST 4413/4409, USA, 555, 555 : 5% BSA 1:500] was added. The cells were protected from light and maintained at room temperature for 1 h. The nuclei were counterstained with DAPI (Vectorlabs, H‐1200‐10, USA). An Olympus VS120‐SL 20‐fold filter (green, blue, and ultraviolet) was used to image samples, and three colours were synthesized.
Ab6326
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab6326 is a primary antibody. It is a rabbit polyclonal antibody that recognizes the target antigen. The antibody is purified and suitable for use in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (25 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (10 mentions)
5 bromo 2 deoxyuridine brdu, by Merck Group (9 mentions)
Mab377, by Merck Group (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
Axio observer z1, by Zeiss (9 mentions)
B5002, by Merck Group (8 mentions)
C6891, by Merck Group (7 mentions)
A11006, by Thermo Fisher Scientific (7 mentions)
Ab15580, by Abcam (6 mentions)
Bromodeoxyuridine (brdu), by Abcam (6 mentions)
Ab18465, by Abcam (6 mentions)
Ab150157, by Abcam (5 mentions)
Ab18723, by Abcam (5 mentions)
Triton x 100, by Merck Group (5 mentions)
Ab290, by Abcam (5 mentions)
Bromodeoxyuridine (brdu), by Nikon (5 mentions)
Ab142567, by Abcam (5 mentions)
Ab5535, by Merck Group (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
306 protocols using ab6326
1
Immunohistochemical Profiling of Muscle Stem Cells
2
Immunohistochemical and Immunocytochemical Analysis of Cell Cycle Markers
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Immunohistochemical staining for H2A histone family, member X (γ-H2AX, Abcam, ab81299), BrdU (Abcam, ab6326), Ki67 (Abcam, ab15580), Cyclin A2 (Abcam, ab181591), Cyclin E1 (Abcam, ab52189) and Cyclin D1 (Abcam, ab134175) were performed on 4% paraformaldehyde-fixed liver sections. 5μm-thick slices were incubated with primary antibodies at 4? overnight followed by biotinylated secondary antibody and the avidin/biotin horseradish peroxidase system (Vectastain DAB Kit; Vector Laboratories, Burlingame, CA). The nuclei were counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO) and the sections were covered in neutral balsam (Solarbio, Beijing, CHN). Immuno-fluorescent staining for β-catenin (Sigma-Aldrich, MAB2081) and PHH3 (Roche, 760-4591) were detected on paraffin-embedded liver section. Slides were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies conjugated with fluorescent dye at 37°C for 30 min.
Immunocytochemical staining for BrdU (Abcam, ab6326) and Ki67(Abcam, ab15580), cells were fixed, permeabilized and blocked, then incubated with primary antibody, followed by fluorescence-tagged secondary antibodies. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO). Images were acquired with a 50i Nikon fluorescence microscope (Nikon, Melville, NY).
Immunocytochemical staining for BrdU (Abcam, ab6326) and Ki67(Abcam, ab15580), cells were fixed, permeabilized and blocked, then incubated with primary antibody, followed by fluorescence-tagged secondary antibodies. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO). Images were acquired with a 50i Nikon fluorescence microscope (Nikon, Melville, NY).
3
Immunofluorescence Staining of Neural Markers
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Immunofluorescence staining was conducted as previously described (13 (link)). Staining was conducted overnight at 4°C with anti-NeuN (1:500; ab104225; Abcam, Waltham, MA), rabbit anti-CD68 (1:500; ab125212; Abcam), rat anti-BrdU (1:500; ab6326; Abcam) and/or rabbit anti-Iba1 (1:500; ab178847; Abcam) primary antibodies. Sections were then incubated with appropriate secondary antibodies for 60 mins at room temperature (1:500; Alexa Fluor goat anti-rabbit 594, A11037; Alexa Fluor goat anti-rabbit 488, A11008; Alexa Fluor goat anti-rat 594, A11007; Invitrogen, Waltham, MA) before mounting with DAPI Fluoromount-G (0100–20; SouthernBiotech, Birmingham, AL). Fluorescent images were captured with either an EVOS fluorescent microscope (ThermoFisher Scientific, Waltham, MA) or BZ-X800E fluorescent microscope (Keyence, Itasca, IL). Image analysis was performed using FIJI (ImageJ/Fiji, version 1.53o; National Institutes of Health, Bethesda, MD).
4
Cell Proliferation and Apoptosis Assays
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SW1088 and SW1783 cell suspensions (3.0 × 104 cells/ml) were plated on 35 mm confocal dishes (Biosharp). Then, bromodeoxyuridine (BrdU) (Roche) with U73122, DMSO, or PBS was added to the medium. After 48 h, cells were fixed with 70% acidic ethanol for 20 min at 4 °C, followed by incubation with primary anti-BrdU antibody (ab6326, Abcam), then Alexa-488 secondary antibody, and finally DAPI. BrdU- and DAPI-positive cells were then counted in ImageJ.
For cell cycle analysis, cells were serum starved for 24 h, and then the medium was replaced with complete growth medium. After 12 or 24 h, cells were collected for DNA staining using propidium iodide (PI), and DNA content was analysed by using a BD Canto II Flow Cytometry System (BD Biosciences) and FlowJo software, v10.6.
For the apoptosis assay, cells were serum starved as described above and then freshly collected and stained for early apoptosis markers using a fluorescein isothiocyanate (FITC) annexin V apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions. Annexin V and PI staining were measured using a flow cytometer and analysed using FlowJo v10.6.
For cell cycle analysis, cells were serum starved for 24 h, and then the medium was replaced with complete growth medium. After 12 or 24 h, cells were collected for DNA staining using propidium iodide (PI), and DNA content was analysed by using a BD Canto II Flow Cytometry System (BD Biosciences) and FlowJo software, v10.6.
For the apoptosis assay, cells were serum starved as described above and then freshly collected and stained for early apoptosis markers using a fluorescein isothiocyanate (FITC) annexin V apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions. Annexin V and PI staining were measured using a flow cytometer and analysed using FlowJo v10.6.
5
Immunofluorescence Staining Protocol
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Tissue sections or adherent cultured cells were incubated for one hour at room temperature in blocking solution (50 mmol/L Tris-HCl, pH 8.0, 0.1 mol/L NaCl, 0.1% Triton X-100, 3% NGS, 0.1% BSA) prior to overnight primary antibody incubation (4°C). Primary antibodies for immunofluorescence studies were: SOX2 (1:1000 dilution, Ab5603, Millipore), Ki67 (1:500 dilution, ab15580, Abcam), BrdU (1:500 dilution, ab6326, Abcam), NeuN (1:500 dilution, ab177487, Abcam), TUJ1 (1:200 dilution, T8660, Sigma-Aldrich), GFAP (1:500 dilution, Z0334, Agilent), MBP (1:300 dilution, SMI-99P, Covance), OLIG2 (1:200 dilution, AB9610, Millipore), OLIG2 (1:50 dilution, MABN50, Millipore), IBA1 (1:200 dilution, ab5076, Abcam), CD31 (1:100 dilution, ab24590, Abcam), ACTA2 (1:400 dilution, ab5694, Abcam), GFP (1:2000 dilution, ab13970, Abcam), CD31 (1:300 dilution, AF3628-SP, R&D), CD146 (1:100 dilution, 134701, BioLegend). We used immunofluorescence staining with Alexa Fluor 488, 555, or 647 (Life Technologies) and biotin-streptavidin-Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch), as well as horseradish peroxidase-based Vectastain ABC Kit (Vector Laboratories). Image acquisitions were performed using a Zeiss LSM880 confocal microscope and image editing done using ZEN, Photoshop or ImageJ.
6
DNA Fiber Analysis of Replication
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DNA fiber analyses were carried out as reported with minor modification (33 (link)). Briefly, cells were first pulse-labeled with 25 μM IdU for 30 min, washed three times with PBS, then pulse-labeled with 250 μM CldU for 30 min followed by 4 hrs of treatment with 2mM HU. Cells were harvested and resuspended in PBS for a final concentration of 1500 cells/μl. Cells were then lysed in lysis buffer (200 mM Tris–HCl (pH 7.5), 50 mM EDTA, 0.5% SDS), DNA fibers were stretched onto positively charged glass slides and fixed in methanol:acetic acid (3:1). After rehydration in PBS twice for 5 min, DNA were denatured with 2.5 M HCl for 1 h, slides were then washed with PBS for three times and blocked with 5% BSA at 37°C for 1 h. The newly replicated ldU and CIdU tracks were immunostained using mouse-anti-BrdU (1/100, 347580, BD Biosciences) and rat-anti-BrdU (1/100, Ab6326, Abcam) primary antibodies, and Alexa Fluor 546 goat-anti-mouse IgG1 (1/500, A-21123, ThermoFisher Scientific) and Alexa Fluor 488 goat-anti-rat (1/500, A-11006, ThermoFisher Scientific) secondary antibodies respectively. Coverslips were mounted using ProLong Gold Antifade Mountant (ThermoFisher Scientific).
7
Corneal Histologic and Molecular Assays
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After clinical examination, rabbits were humanely killed with an intravenous injection of potassium chloride (1 mg/kg) under deep anesthesia, and corneas were extracted. The half of a cornea was subjected to histologic assays, and another half to molecular assays.
For histologic examination, the frozen sections were subjected to hematoxylin-eosin staining, immunostaining for BrdU, p63, and CK (cytokeratin) 3/12. For detection of BrdU-labelled nuclei, rat mAb to BrdU (1 : 100, ab6326, Abcam) was used. For p63 and CK 3/12 immunostaining, goat mAb to rabbit p63 (1 : 100, ab124762, Abcam), and mouse mAb to rabbit CK3/12 (1 : 100, ab68260, Abcam, Cambridge, UK) were used as primary antibodies. Secondary antibodies used were goat anti-rat IgG, TRITC (Millipore, Billerica Massachusetts 01821, USA) for BrdU and Ck3/12, and goat anti-mouse IgG Alexa 488 (Invitrogen, Waltham, MA, USA) for p63. Nuclei were counterstained using Hoechst 33342 (Sigma, St. Louis, MO, USA).
The stained slides were observed under a fluorescent microscope (BX-61, Olympus, Melville, NY, USA) with ×200 magnification. The number of positively stained cells was counted in three different sections of each eye, and the average count was determined.
For histologic examination, the frozen sections were subjected to hematoxylin-eosin staining, immunostaining for BrdU, p63, and CK (cytokeratin) 3/12. For detection of BrdU-labelled nuclei, rat mAb to BrdU (1 : 100, ab6326, Abcam) was used. For p63 and CK 3/12 immunostaining, goat mAb to rabbit p63 (1 : 100, ab124762, Abcam), and mouse mAb to rabbit CK3/12 (1 : 100, ab68260, Abcam, Cambridge, UK) were used as primary antibodies. Secondary antibodies used were goat anti-rat IgG, TRITC (Millipore, Billerica Massachusetts 01821, USA) for BrdU and Ck3/12, and goat anti-mouse IgG Alexa 488 (Invitrogen, Waltham, MA, USA) for p63. Nuclei were counterstained using Hoechst 33342 (Sigma, St. Louis, MO, USA).
The stained slides were observed under a fluorescent microscope (BX-61, Olympus, Melville, NY, USA) with ×200 magnification. The number of positively stained cells was counted in three different sections of each eye, and the average count was determined.
8
Immunohistochemical Analysis of Intestinal Tissues
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Paraffin embedded sections of intestine from wt and
Crh−/− mice were processed according to the
manufacturer’s protocol. Briefly, sections were deparaffinized,
washed and antigen retrieved with 10 mM citrate buffer (pH 6.3). The
tissues were then washed, covered with blocking buffer (20 mM HEPES,
1% bovine serum albumin (BSA), 135 mM NaCl) for
5 minutes and incubated overnight with the primary antibody (LC3,
1:2000, PM036, MBL; Crh, 1:100, NBP1-35703, Novus Biologicals; F4/80, 1:100,
ab6640, Abcam; CD11b, 1:50, 553307, BD Pharmingen) diluted in blocking buffer.
For BrdU immunohistochemistry (1:50 anti-BrdU antibody, ab6326, Abcam),
following antigen retrieval, sections were sequentially incubated in 2N HCl for
30 minutes at 37 °C, in 0.1 M
borate buffer for 10 min and in blocking solution (10% Normal Goat
Serum). Finally, sections were washed and incubated with a secondary antibody
(1:500 Alexa Fluor® 488 Goat Anti – rabbit IgG,
Invitrogen) and mounted with VECTASHIELD® Mounting Medium with DAPI
(H-1200, Vector Laboratories). F4/80/CD11b
Crh−/− mice were processed according to the
manufacturer’s protocol. Briefly, sections were deparaffinized,
washed and antigen retrieved with 10 mM citrate buffer (pH 6.3). The
tissues were then washed, covered with blocking buffer (20 mM HEPES,
1% bovine serum albumin (BSA), 135 mM NaCl) for
5 minutes and incubated overnight with the primary antibody (LC3,
1:2000, PM036, MBL; Crh, 1:100, NBP1-35703, Novus Biologicals; F4/80, 1:100,
ab6640, Abcam; CD11b, 1:50, 553307, BD Pharmingen) diluted in blocking buffer.
For BrdU immunohistochemistry (1:50 anti-BrdU antibody, ab6326, Abcam),
following antigen retrieval, sections were sequentially incubated in 2N HCl for
30 minutes at 37 °C, in 0.1 M
borate buffer for 10 min and in blocking solution (10% Normal Goat
Serum). Finally, sections were washed and incubated with a secondary antibody
(1:500 Alexa Fluor® 488 Goat Anti – rabbit IgG,
Invitrogen) and mounted with VECTASHIELD® Mounting Medium with DAPI
(H-1200, Vector Laboratories). F4/80/CD11b
9
Immunochemical Identification of CNS Cell Types
10
Quantifying Pancreatic Cell Populations
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After antigen retrieval, deparaffinized pancreatic tissue sections were blocked with normal donkey serum and immunostained with anti-insulin (N1542; Dako North America, Carpinteria, CA), anti-glucagon (PA1–85465; Pierce Chemical Company, Rockford, IL), anti-GPR43 (PAB16402; Abnova), or anti-BrdU (ab6326; Abcam), followed by incubation with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 and counterstained with DAPI. For β-cell mass analysis, images were captured using a Hamamatsu NanoZoomer HT-2.0 slide scanner and were analyzed using Image-Pro Plus software. A minimum of five slides per animal, 100 μm apart, were used to quantify the relative pancreatic β-cell area, by dividing the total pancreatic area by the insulin-positive (Ins+) area. The percentage of proliferating β-cells was calculated by dividing the number of BrdU-positive (BrdU+) and Ins+ cells by the number of Ins+ cells.
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