Chromosome Spreading and Immunofluorescence Analysis

Spermatocyte and oocyte chromosome spreading was performed as previously described (24 (link)). Produced antibody used for western blotting: Mouse anti-SPIDR polyclonal antibody was prepared against mouse SPIDR protein by Dai-an Biological Technology Incorporation (Wuhan, China). Primary antibodies used for immunofluorescence were as following: rabbit anti-SYCP1 (1:500 dilution; Abcam #ab15090), mouse anti-SYCP3 (1:500 dilution; Abcam #ab97672), rabbit anti-RPA2 (1:200 dilution; Abcam #ab76420), rabbit anti-RAD51 (1:200 dilution; Thermo Fisher Scientific #PA5-27195), rabbit anti-DMC1 (1:200 dilution; homemade); mouse anti-γH2AX (pSer139) (1:1000 dilution; Millipore #05–636), mouse anti-MLH1 (1:100 dilution; BD Biosciences #550838), rabbit anti-BLM (1:200 dilution; Invitrogen #PA5-27384), rabbit anti-MSH4(1:200 dilution; Abcam #ab58666); rat anti-HEI10 (1:100 dilution; homemade); Rabbit anti-MSY2(1:200 dilution; Abcam #ab33164). Primary antibodies were detected with Alexa Fluor 488- or 594-conjugated secondary antibodies (1:500 dilution; Abcam #ab150080, #ab150077, #ab150113 and #ab150116) for 1 h at room temperature. The slides were mounted using Mounting Medium with DAPI (Abcam #ab104139).

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Huang T., Wu X., Wang S., Bao Z., Wan Y., Wang Z., Li M., Yu X., Lv Y., Liu Z., Chen X., Chan W.Y., Gao F., Lu G., Chen Z.J, & Liu H. (2023). SPIDR is required for homologous recombination during mammalian meiosis. Nucleic Acids Research, 51(8), 3855-3868.

Publication 2023

ab15090 ab97672 ab76420 ab58666 ab150080 ab150116 ab104139

Alexa fluor 488 Anti antibody Antibodies Antibody Biological Chromosome Dapi Dilution Dmc1 Hei10 Immunofluorescence Mlh1 Mouse Mouse spidr protein Msh4 Oocyte Rabbit Rpa2 Spermatocyte

Corresponding Organization : Chinese Academy of Medical Sciences & Peking Union Medical College

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Variable analysis

independent variables

Chromosome spreading technique for spermatocytes and oocytes

dependent variables

Localization and expression of proteins involved in meiosis, such as SYCP1, SYCP3, RPA2, RAD51, DMC1, γH2AX, MLH1, BLM, MSH4, HEI10, and MSY2

control variables

Primary antibody dilutions for immunofluorescence (e.g., 1:500, 1:200, 1:100, 1:1000)
Secondary antibody dilution (1:500)
Mounting medium with DAPI

positive controls

Homemade rabbit anti-DMC1 antibody
Homemade rat anti-HEI10 antibody

negative controls

Not explicitly mentioned

Annotations

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