Hermaphrodites were staged, anesthetized and dissected as described for sperm/oocyte staining. Gonads were fixed in 2% paraformaldehyde (PFA) in 100 nM pH 7.2 K2PO4 for 30 minutes, rocking at RT. Gonads were washed 2x in PBST: first wash quick and second wash for 5 minutes at RT. Gonads were fixed in methanol for 30 minutes at −20°C. Gonads were washed 2x, following the same procedure as after the PFA fix. Gonads were blocked for 1 hour, rocking at RT. Primary antibodies—mouse α-V5 (Bio-Rad), rat α-OLLAS (Novis, NBP1-06713), rabbit α-ERK (Santa Cruz Biotechnology, Sc94)—were diluted 1:1000, 1:200, 1:1000 in blocking solution, respectively. Gonads incubated with primary antibodies overnight at 4°C. Gonads were then washed 2x PBST quickly and 2x PBST for 10 minutes. Secondary α-mouse alexa555, α-rat alexa647, and a-rabbit alexa488 antibodies, and DAPI (1 μg/mL) were all diluted 1:1000 in block solution and incubated for 2 hours, rocking in the dark at RT. Secondary antibodies were removed and gonads were washed 4x in PBST—2x quickly and 2x for 10 minutes in the dark. Gonads were mounted in 18 μL Prolong Glass antifade, sealed with VALAP, and stored at −20°C until imaged.
Mouse α v5
Manufactured by Bio-Rad
Mouse α-V5 is a monoclonal antibody that specifically recognizes the V5 epitope, a synthetic peptide derived from the P and V proteins of the paramyxovirus SV5. This antibody can be used for the detection and purification of recombinant proteins containing the V5 tag.
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2 protocols using mouse α v5
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Immunostaining of Hermaphrodite Gonads
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Immunostaining of Hermaphrodite Gonads
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Hermaphrodites were staged, anesthetized and dissected as described for sperm/oocyte staining. Gonads were fixed in 2% paraformaldehyde (PFA) in 100 nM pH 7.2 K2PO4 for 30 minutes, rocking at RT. Gonads were washed 2x in PBST: first wash quick and second wash for 5 minutes at RT. Gonads were fixed in methanol for 30 minutes at −20°C. Gonads were washed 2x, following the same procedure as after the PFA fix. Gonads were blocked for 1 hour, rocking at RT. Primary antibodies—mouse α-V5 (Bio-Rad), rat α-OLLAS (Novis, NBP1-06713), rabbit α-ERK (Santa Cruz Biotechnology, Sc94)—were diluted 1:1000, 1:200, 1:1000 in blocking solution, respectively. Gonads incubated with primary antibodies overnight at 4°C. Gonads were then washed 2x PBST quickly and 2x PBST for 10 minutes. Secondary α-mouse alexa555, α-rat alexa647, and a-rabbit alexa488 antibodies, and DAPI (1 μg/mL) were all diluted 1:1000 in block solution and incubated for 2 hours, rocking in the dark at RT. Secondary antibodies were removed and gonads were washed 4x in PBST—2x quickly and 2x for 10 minutes in the dark. Gonads were mounted in 18 μL Prolong Glass antifade, sealed with VALAP, and stored at −20°C until imaged.
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