Rosette leaf material was harvested and directly fixed in ice-cold Carnoy’s fixative (3:1 ethanol/acetic acid) and kept at –20° until use. Nuclei spread preparations for microscopic analysis were made essentially as described by Schubert et al. (2001 (link)) and Tessadori et al. (2009 (link)) using modified enzymatic cell wall–degrading mixture [cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa) in 10 mM citrate buffer, pH 4.5] to digest cell walls. The air-dried slides were mounted in Vectashield (Vector Laboratories) with DAPI (2 µg ml-1) before observation and capturing.
For the (de)etiolation experiments, cotyledons of 5-d-old seedlings were fixed in 4% paraformaldehyde for 3 hr under white light condition or under a safe green light for the dark-grown seedlings, and treated with a solution containing 0.5% cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa), and 0.1% Triton X-100 for 1 hr. Cotyledons from at least three seedlings were isolated and squashed on a glass slide, flash frozen in liquid nitrogen, and incubated with PEMSB (50 mM Pipes, pH 7.3; 5 mM EGTA, pH 7.1; 5 mM MgSO4; 0.05% saponin; 5% wt/vol BSA) before being mounted with Vectashield (Vector Laboratories) supplemented with 2 μg/ml DAPI before observation and capturing.
For the (de)etiolation experiments, cotyledons of 5-d-old seedlings were fixed in 4% paraformaldehyde for 3 hr under white light condition or under a safe green light for the dark-grown seedlings, and treated with a solution containing 0.5% cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa), and 0.1% Triton X-100 for 1 hr. Cotyledons from at least three seedlings were isolated and squashed on a glass slide, flash frozen in liquid nitrogen, and incubated with PEMSB (50 mM Pipes, pH 7.3; 5 mM EGTA, pH 7.1; 5 mM MgSO4; 0.05% saponin; 5% wt/vol BSA) before being mounted with Vectashield (Vector Laboratories) supplemented with 2 μg/ml DAPI before observation and capturing.
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