Immunostaining of dissected gonads was carried out as described (Colaiacovo et al., 2003 ; Saito et al., 2009 ). Worms were permeabilized on Superfrost+slides for 2 min with methanol at −20° and fixed for 30 min in 4% paraformaldehyde in PBS. After blocking with 1% BSA in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature, slides were incubated with primary antibody for 1 h at room temperature. After incubation with fluorescent secondary antibody 1 h at room temperature, slides were DAPI‐stained for 10 min at 500 ng/ml, destained 1 h in PBST, and mounted with Vectashield (Vector Laboratories). The primary antibodies used were as follows: rabbit α‐LAB‐1 (1:200, (de Carvalho et al., 2008 )), rabbit α‐RAD‐51 (1:10,000, SDIX), mouse α‐MAPK‐YT (1:500, M8159; Sigma), rabbit α‐SYP‐2 (1:200, a kind gift from S. Smolikove), rabbit α‐pH3 (D5692, 1:1000; Sigma), and guinea pig α‐HTP‐3 (1:200, (Goodyer et al., 2008 )). The secondary antibodies used were Cy2‐donkey anti‐rabbit, Cy3‐donkey anti‐guinea pig, Cy3‐goat anti‐rabbit, and Cy3‐goat anti‐mouse (all used at 1:500 dilution; Jackson ImmunoResearch Laboratories).
Mouse α mapk yt
Manufactured by Merck Group
Mouse α‐MAPK‐YT is a laboratory reagent used for the detection and quantification of the alpha-isoform of mitogen-activated protein kinase (MAPK) in mouse samples. It is designed for use in various analytical techniques, such as Western blotting and ELISA.
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2 protocols using mouse α mapk yt
1
Immunostaining of C. elegans Gonads
2
Immunostaining Dissected Gonads in Worms
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Immunostaining of dissected gonads was carried out as described (Colaiacovo et al. 2003; Saito et al. 2009) . Worms were permeabilized on Superfrost+ slides for 2 min with methanol at -20° and fixed for 30 min in 4% paraformaldehyde in PBS. After blocking with 1% BSA in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature, slides were incubated with primary antibody for 1 h at room temperature.
After incubation with fluorescent secondary antibody 1 h at room temperature, slides were DAPI stained for 10 min at 500 ng/ml, destained 1 h in PBST, and mounted with Vectashield (Vector Laboratories). The primary antibodies used were as follow: rabbit α-LAB-1 (1:200, (de Carvalho et al. 2008 )), rabbit α-RAD-51 (1:10,000, SDIX), mouse α-MAPK-YT (1:500, M8159; Sigma), rabbit α-SYP-2 (1:200, a kind gift from S. Smolikove), rabbit α-pH3 (D5692, 1:1000; Sigma), and guinea pig α-HTP-3 (1:200, (Goodyer et al. 2008) ). The secondary antibodies used were Cy2-donkey anti-rabbit, Cy3-donkey anti-guinea pig, Cy3-goat anti-rabbit, Cy3-goat anti-mouse (all used at 1:500 dilution; Jackson ImmunoResearch Laboratories).
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 29, 2020. ; https://doi.org/10.1101/2020.10.29.360693 doi: bioRxiv preprint
After incubation with fluorescent secondary antibody 1 h at room temperature, slides were DAPI stained for 10 min at 500 ng/ml, destained 1 h in PBST, and mounted with Vectashield (Vector Laboratories). The primary antibodies used were as follow: rabbit α-LAB-1 (1:200, (de Carvalho et al. 2008 )), rabbit α-RAD-51 (1:10,000, SDIX), mouse α-MAPK-YT (1:500, M8159; Sigma), rabbit α-SYP-2 (1:200, a kind gift from S. Smolikove), rabbit α-pH3 (D5692, 1:1000; Sigma), and guinea pig α-HTP-3 (1:200, (Goodyer et al. 2008) ). The secondary antibodies used were Cy2-donkey anti-rabbit, Cy3-donkey anti-guinea pig, Cy3-goat anti-rabbit, Cy3-goat anti-mouse (all used at 1:500 dilution; Jackson ImmunoResearch Laboratories).
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 29, 2020. ; https://doi.org/10.1101/2020.10.29.360693 doi: bioRxiv preprint
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