Aug 13, 2025
IMMUNOFLUORESCENCE PROTOCOL
- Yuanzhuo Zhou1,2,
- Kohei Asai1,2,
- Hirohisa Kyogoku1,3,
- Tomoya S. Kitajima1,2
- 1Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan;
- 2Graduate School of Biostudies, Kyoto University, Kyoto, Japan;
- 3Graduate School of Agricultural Science, Kobe University, Kobe, Japan
- Designing protein-based artificial kinetochore
Protocol Citation: Yuanzhuo Zhou, Kohei Asai, Hirohisa Kyogoku, Tomoya S. Kitajima 2025. IMMUNOFLUORESCENCE PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46joogo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 13, 2025
Last Modified: August 13, 2025
Protocol Integer ID: 224589
Keywords: staining of mouse oocyte, immunofluorescence protocol, immunofluorescence protocol this protocol, mouse oocyte, paraformaldehyde solution
Abstract
This protocol describes the fixation and
immunofluorescent staining of mouse oocyte in paraformaldehyde solution.
Materials
1.6% formaldehyde, 100 mM PIPES (pH 7.0), 1 mM MgCl2, 0.1% Triton X-100, PBS, 3% BSA, primary antibodies, secondary antibodies, Hoechst 33342 (Invitrogen)
Troubleshooting
Protocol
Prepare fixation buffer consisting of 1.6% formaldehyde in 100 mM PIPES (pH 7.0), 1 mM MgCl2, and 0.1% Triton X-100.
Fix oocytes in fixation buffer at room temperature for 30 minutes.
Wash oocytes 4 times in PBS containing 0.1% Triton X-100 (PBT).
Incubate oocytes in PBT at 4 °C overnight.
Prepare blocking solution PBT containing 3% BSA.
Block oocytes in blocking solution at room temperature for 1 hour.
Prepare primary antibody solution in 3% BSA-PBT.
Incubate oocytes with primary antibodies at 4 °C overnight.
Wash oocytes 4 times in 3% BSA-PBT.
Prepare secondary antibody solution in 3% BSA-PBT containing 5 µg/ml Hoechst 33342 (Invitrogen).
Incubate oocytes in secondary antibody solution at room temperature for 2 hours or at 4 °C overnight.