Meiotic surface spreads of prophase I female meiocytes were prepared using an adaptation (Reinholdt et al., 2004 (link)) of a drying-down technique (Peters et al., 1997 ) that was described in great detail in the former reference. Meiotic stages (leptonema-diakinesis) were determined based on SYCP3 staining patterns (Gray and Cohen, 2016 (link)). Slides were stored at −80°C until immunostained. For staining, slides were brought to room temperature (RT) and washed once with PBS+0.1% Tween-20 (PBS-T). Slides were blocked for 40 minutes at RT with PBS-T containing 5% normal goat serum (5%GS-PBS-T). Primary antibodies were diluted into 5%GS-PBS-T and incubated overnight at RT in a humidified chamber. Antibodies and dilutions used included: rabbit anti-RAD51 (1:250 Abcam 176458), mouse anti-SYCP3 (1:600 Abcam) and guinea pig anti-HORMAD2 antibody (1:1000, kind gift from Attila Toth). Secondary antibodies used were diluted 1:1000 in in 5%GS-PBS-T and included goat anti-rabbit Alexa 488/594, goat anti-mouse Alexa 488/594 and goat anti-guinea pig Alexa 488/594. Images were taken using an Olympus microscope with 40× lens or 100× immersion oil lens and CCD camera.
Mouse anti sycp3
Manufactured by Abcam
Mouse anti-SYCP3 is a primary antibody that recognizes the SYCP3 protein. SYCP3 is a component of the synaptonemal complex, which is involved in chromosome organization and segregation during meiosis. This antibody can be used for various applications, such as immunohistochemistry and Western blotting, to detect and study the SYCP3 protein.
Automatically generated - may contain errors
Lab products found in correlation
Mouse whole chromosome painting probes for chromosome 18, by Zeiss (2 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Rabbit anti histone h3, by Abcam (1 mentions)
Mouse anti gapdh, by ABclonal (1 mentions)
Rabbit anti mouse igg h l hrp, by Abcam (1 mentions)
4 protocols using mouse anti sycp3
1
Meiotic Surface Spread Immunostaining
2
Fluorescent Chromosome Painting Assay
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Slides were washed briefly in PBS, 2 × SSC and then denatured in 70% formamide in 2 × SSC for 30 min at 80 °C. Ten microliters of fluorescently labelled Mouse Whole Chromosome Painting Probes for chromosome 18 or chromosome 19 (Carl Zeiss Ltd.) were denatured at 70 °C for 5 min, re-annealed for 15 min at 37 °C and hybridised under a coverslip overnight at 37 °C. Slides were then washed four times in 2 × SSC for 3 min at 45 °C each, four times in 0.1 × SSC for 3 min at 60 °C each, and then with 2 × SSC/0.1% Tween-20 for 3 min at room temperature. Axial elements were then stained with 1:500 mouse anti-SYCP3 (Abcam, Online Resource 1 ) as described for immunostaining meiotic chromosome spreads. Chromosome domains were judged either to be separate if there were two domains visible that shared no overlap, or overlapping if there was a single continuous domain.
3
Protein Extraction and Western Blotting of Mouse Testicular Tissue
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Mouse testicular tissue was lysed using nuclear and cytoplasmic protein extraction kit (Beyotime, P0027) according to the manufacturer’s instructions. The lysates were ultracentrifuged at 12000 g for 10 min at 4 °C. The supernatants and remaining sediment were collected separately. The concentration of protein in the sediment and supernatants were measured using a bicin-choninic acid assay (Beyotime Biotechnology, P0012S). For western blot assay, 50 μg of protein samples was loaded on 10% SDS-PAGE gel and runed 1.5 h at 100 V before transferring to PVDF membranes. The antibodies used were as follows. Rabbit anti-CDKN2AIP (1:1000, Proteintech, Cat No.16615-1-AP), Mouse anti-SYCP3 (1:1000, Abcam, Cat No. ab97672), Rabbit anti-histone H3(1:1000, Abcam, Cat No.ab1791), Rabbit anti-PRM1(1:1000, Affinity, Cat No.DF5045), Rabbit anti PRM2(1:1000, Proteintech, Cat No.14500-1-AP), Rabbit anti-SUN1(1:1000, Abcam, Cat No.ab103021), Mouse anti-GAPDH (1:10,000, ABclonal, Cat No.AC002), Goat Anti-Rabbit IgG H&L (HRP) (1:8000, Abcam, ab6721), Rabbit Anti-Mouse IgG H&L (HRP) (1:8000, Abcam, ab6728).
4
Chromosome Painting and SYCP3 Immunostaining
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