Gravid hermaphrodites were picked into 5 µl of M9 buffer onto a poly-lysine coated slide (Sigma Aldrich). A coverslip was placed on top and worms were gently compressed to allow embryos to extrude. Embryos were immediately freeze-cracked in liquid nitrogen and fixed with methanol for 10 min followed by acetone for an additional 10 min. After 3 washes in PBS containing 0.25 % Triton X-100 (PBS-T), slides were blocked in PBS-T with 0.5 % BSA before overnight incubation with mouse monoclonal primary antibody diluted 1/200 (PIIA3 against YP17021 (link), a kind gift of Susan Strome) at 4 ° C. The slides were then washed with PBS-T, and incubated for 2 h with Alexa-488 anti-mouse secondary antibody (Invitrogen) at room temperature. After 3 washes in PBS-T, the samples were mounted in Fluoroshield with DAPI mounting medium (Sigma Aldrich). Stained embryos were imaged by epifluorescence microscopy. Quantification was performed using ImageJ. Data were analysed by generalized linear model analysis with the ‘lme4’ package in R, with the following terms included in the model: maternal age, embryo nuclei count as fixed effects and staining replicate and mother worm as random effects.
Poly lysine coated slide
Manufactured by Merck Group
The Poly-lysine coated slide is a type of laboratory equipment used for various applications in cell biology and microscopy. The slide is coated with a layer of poly-L-lysine, a synthetic polypeptide that enhances the adhesion of cells, tissues, or other biological samples to the surface of the slide. This feature allows for improved sample attachment and analysis under a microscope.
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Lab products found in correlation
2 protocols using poly lysine coated slide
1
Immunostaining of C. elegans Embryos
2
Immunostaining of C. elegans Embryos
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Gravid hermaphrodites were picked into 5 µl of M9 buffer onto a poly-lysine coated slide (Sigma Aldrich). A coverslip was placed on top and worms were gently compressed to allow embryos to extrude. Embryos were immediately freeze-cracked in liquid nitrogen and fixed with methanol for 10 min followed by acetone for an additional 10 min. After 3 washes in PBS containing 0.25 % Triton X-100 (PBS-T), slides were blocked in PBS-T with 0.5 % BSA before overnight incubation with mouse monoclonal primary antibody diluted 1/200 (PIIA3 against YP17021 (link), a kind gift of Susan Strome) at 4 ° C. The slides were then washed with PBS-T, and incubated for 2 h with Alexa-488 anti-mouse secondary antibody (Invitrogen) at room temperature. After 3 washes in PBS-T, the samples were mounted in Fluoroshield with DAPI mounting medium (Sigma Aldrich). Stained embryos were imaged by epifluorescence microscopy. Quantification was performed using ImageJ. Data were analysed by generalized linear model analysis with the ‘lme4’ package in R, with the following terms included in the model: maternal age, embryo nuclei count as fixed effects and staining replicate and mother worm as random effects.
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