Fetal oocyte cytospreads were performed as previously described [49 (link)]. Slides were incubated with the first antibody against rabbit/mouse SCP3 (rabbit polyclonal, Novus Biologicals Littleton, NB300-232, USA; or mouse polyclonal, Abcam, ab97672, USA), γH2AX (mouse polyclonal, Abcam, ab26350) or RAD51 (rabbit polyclonal, Abcam, ab133534), for 8 h at 37 °C. After overnight blocking with TBS supplemented with 10% goat serum (Boster, AR009, Wuhan, China), Cy3-labeled goat anti-rabbit (Beyotime, A0516) were used to label SCP3/RAD51 and FITC-conjugated goat anti-mouse antibodies (Beyotime, A0568), were used to label γH2AX/SCP3 at 1:100 dilution for 30 min at 37 °C in the dark; DNA was stained with Hoechst 33342. Pictures were taken with an Olympus fluorescence microscope (BX51).
Cy3 labeled goat anti rabbit
Manufactured by Beyotime
Sourced in China
Cy3-labeled goat anti-rabbit is a secondary antibody used in various immunological techniques, such as immunofluorescence and Western blotting. It is produced by conjugating the Cy3 fluorescent dye to goat-derived antibodies that specifically recognize and bind to rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Ab133534, by Abcam (1 mentions)
Ab97672, by Abcam (1 mentions)
Ab26350, by Abcam (1 mentions)
Ar009, by Boster Bio (1 mentions)
Nb300 232, by Abcam (1 mentions)
A0568, by Beyotime (1 mentions)
A0516, by Beyotime (1 mentions)
Fitc labeled goat anti mouse, by Beyotime (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Cy3 labeled goat anti mouse, by Beyotime (1 mentions)
5 protocols using cy3 labeled goat anti rabbit
1
Immunostaining of Meiotic Chromosome Proteins
2
Kidney Immunofluorescence Staining
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Immunofluorescence staining of the kidneys was performed on paraffin sections. After fixation and antigen retrieval, nonspecific binding was blocked with 3% BSA. Tissue sections were incubated with the mixed primary antibodies of rabbit anti-Ki67 (1:400, Cat# 9129, CST, USA) and mouse anti-Ecadherin (1:300, Cat# 14472, Abcam, USA) overnight at 4°C. After washed three times with PBS, the tissue sections were incubated with mixed secondary antibodies of Cy3-labeled goat anti-rabbit (1:500, Cat# A0516, Beyotime, China) and FITC-labeled goat anti-mouse (1:500, Cat# A0568, Beyotime, China). After another washing with PBS, the sections were coverslipped with fluorescent mounting medium with DAPI (4,6-diamidino-2-phenylindole) (Cat# ZLI-9557, ZSGB-BIO, China). The staining was examined using fluorescence microscope (Leica, Germany). All images were acquired using the same microscope and camera set. Six to ten high-power fields from the outer medulla and cortex in each kidney examined were captured, then the number of Ki67-positive cells was counted, and the fluorescence intensity of Ecadherin was measured by Image Pro Plus (Media Cybernetics, USA).
3
Peritoneal Tight Junction Protein Analysis
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Immunofluorescence was carried out to detect the distribution patterns of claudin-1, occludin, ZO-1 and JAM-1 proteins in the peritoneum. Thin sections (3 mm) of paraffin-embedded sections of peritoneum were prepared and mounted into adhesive microscopic glass slides. After dewaxing, the sections were permeabilized with citrate buffer for 15 min in microwave, washed 3 times with phosphate buffered saline (PBS) and then blocked with 5% GSA (diluted in PBS) for 1.0 h at RT. The sections were incubated overnight with rabbit anti-occludin, anti-ZO-1, anti-claudin-1, and anti-JAM-1 antibody, respectively, at a 1:100 dilution at 4 °C, then incubated with Cy3-labeled goat anti rabbit (Beyotime, Shanghai, China) at a dilution of 1:100 for 1 h at RT. Fluorescence images were captured using a confocal microscope. The Image J software 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was used to evaluate the amounts of each protein present at the intercellular junctions by semi-quantitatively measuring fluorescence density in the selected areas.
4
Quantitative Analysis of Tight Junction Proteins in Aorta
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The distribution patterns of claudin-1, occludin, ZO-1, and JAM-1 proteins in aorta were detected by immunofluorescence. Thin sections of paraffin-embedded aorta were prepared and mounted into adhesive microscopic glass slides. The sections were dewaxed and permeabilized with citrate buffer for 15 min in microwave, washed, and blocked with 5% GSA (diluted in phosphate buffered saline) at room temperature for 1 h. After incubating with rabbit anti-claudin-1, anti-occludin, anti-ZO-1, and anti-JAM-1 antibody (diluted at a 1:100), respectively, at 4°C overnight, the sections were incubated with Cy3-labeled goat anti-rabbit (diluted at 1:100, Beyotime, China) at room temperature for 1 h. A confocal microscope was used to capture fluorescence images (magnification 10 × 40). Image J software version 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was used to semiquantitatively measure fluorescence density in the selected areas to assess the amounts of each TJ protein (35 (link)). The image analyses were performed in a blinded manner. The analyst was unaware of how the animal was treated prior to tissue sectioning.
5
Double Immunofluorescence Staining Protocol
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Protocols for double IF staining were similar to IF-TSA staining except for the following steps. After sections were stained with FITC-labeled tyramide for PGP9.5, TH or VIP, sections were incubated, respectively, with mouse anti-K7 (ZSGB-BIO, ZM0071, Beijing, China), rabbit ani-S100P (Novus, NBP1-95671, MO, USA), or rabbit ani-K14 (ZSGB-BIO, ZA0540, Beijing, China) for 2 h at room temperature in the dark, followed by incubation with Cy3-labeled goat anti-rabbit (Beyotime, A0516, Jiangsu, China) or Cy3-labeled goat anti-mouse (Beyotime, A0521, Jiangsu, China) secondary antibodies for 10 min at room temperature in the dark.
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