Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes
Abstract
:1. Background
2. Method
- Under a dissecting microscope, place 100 microlitres (μL) of water on a Labtek slide. Dissect 100–120 anthers (on to the water droplet).Comments: To remove water or any other solution, use a fine-tip needle.
- Transfer 50 μL of 4% paraformaldehyde fixative solution onto the dissected anthers.
- Fix the anthers for 60–80 min on ice.
- Wash the anthers three times, 5 min each, with Buffer A.Comments: After three washes, aggregate the anthers and quickly add water. Aspirate the water with the help of syringe. Repeat this step two more times (5 min each). Remember to keep 5 μL of water with the sample during each wash to prevent it from drying.
- Remove the wash buffer and add 10–20 μL of water to the anthers. Cover the anthers with a coverslip.
- Gently press on the coverslip using the tip of the fine forceps to release the meiocytes. Make sure the coverslips do not break.Comment: Hard press will break the coverslip, which will be critical to collect the released meiocytes.
- Add 5–10 μL of water to one side of the coverslip and collect the solution that contains meiocytes from the other side of the coverslip using a fine pipette tip.
- Transfer the meiocytes onto a new poly-L-lysine-coated slide and incubate on dry ice for 10–15 min.
- Thaw the slide to room temperature.
- Transfer 5 μL of the meiocyte solution onto each of the 5–10 new coated slides and cover the cells with a coverslip.
- Set the slides with coverslips precisely under folder paper towel and squash the sample area with the thumb. This step should be deliberately performed as increasing pressure has a propensity to break the chromosomes.
- Freeze the slide at −80 °C freezer with coverslips hanging outside the edge of the slide.
- Remove the coverslip while still frozen and dry the slides at room temperature.
- Soak the sample with 500 μL of cell permeabilization solution at room temperature with/without agitation for 30 min.
- Wash slides twice with 500 μL of wash buffer for 5 min each wash.
- Block the sample in 200 μL of blocking buffer for 1 h.
- Dilute primary antibody (to 1000-fold) in blocking buffer.
- Apply 100 μL of diluted antibody to the sample area and put parafilm.
- Incubate the slides in a slide incubation box at 4 °C overnight.
- Next day, wash thrice with wash buffer and make sure the slides do not dry out.
- Apply the secondary antibody (to 1000-fold) diluted in blocking solution to the sample area and cover it with parafilm.
- Incubate overnight at 4 °C in the slide incubation box.
- Wash the secondary antibody with wash buffer three times and semidry the slides.
- Apply 5 μL of 4’,6-diaminido-2-phenlyinidole (DAPI) in Vectashield on the sample area and put coverslip for image acquisition.
3. Materials
3.1. Plants
3.2. Tools and Equipment
- Dry ice and −80 °C refrigerator
- Coverslips and poly-L-lysine-coated slides
- Slide incubation box with a wet paper towel to keep proper humidity
- Dumont Forceps with long, fine tips
- Commercially available water repellent slides, such as the Lab-Tek™ Chamber Slide™ System (Thermo Fisher Scientific, Waltham, MA, USA)
- Stereomicroscope, florescence microscope, and image acquisition software
3.3. Solutions Recipe
4. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
- Yang, X.; Yuan, L.; Makaroff, C.A. Immunolocalization Protocols for Visualizing Meiotic Proteins in Arabidopsis thaliana: Method 3. Methods Mol. Biol. 2013, 990, 109–118. [Google Scholar] [PubMed]
- Cai, X.; Dong, F.; Edelmann, R.E.; Makaroff, C.A. SYN1 cohesin protein is required for sister chromatid arm cohesion and homologous chromosome pairing. J. Cell Sci. 2003, 116, 2999–3007. [Google Scholar] [CrossRef] [PubMed]
- Higgins, J.D.; Sanchez-Moran, E.; Armstrong, S.J.; Jones, G.H.; Franklin, F.C.H. The Arabidopsis synaptonemal complex protein ZYP1 is required for chromosome synapsis and normal fidelity of crossing over. Genes Dev. 2005, 19, 2488–2500. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Armstrong, S.J.; Caryl, A.P.; Jones, G.H.; Franklin, F.C.H. Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica. J. Cell Sci. 2002, 115 Pt 18, 3645–3655. [Google Scholar] [CrossRef] [PubMed] [Green Version]
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De, K.; Yuan, L.; Makaroff, C.A. Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes. Plants 2018, 7, 93. https://doi.org/10.3390/plants7040093
De K, Yuan L, Makaroff CA. Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes. Plants. 2018; 7(4):93. https://doi.org/10.3390/plants7040093
Chicago/Turabian StyleDe, Kuntal, Li Yuan, and Christopher A. Makaroff. 2018. "Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes" Plants 7, no. 4: 93. https://doi.org/10.3390/plants7040093