ImmunoFISH: Simultaneous Visualisation of Proteins and DNA Sequences Gives Insight Into Meiotic Processes in Nuclei of Grasses

Frontiers in Plant Science
Adél SepsiTrude Schwarzacher

Abstract

ImmunoFISH is a method combining immunolabelling (IL) with fluorescent in situ hybridisation (FISH) to simultaneously detect the nuclear distribution of proteins and specific DNA sequences within chromosomes. This approach is particularly important when analysing meiotic cell division where morphogenesis of individual proteins follows stage-specific changes and is accompanied by a noticeable chromatin dynamism. The method presented here is simple and provides reliable results of high quality signal, low background staining and can be completed within 2 days following preparation. Conventional widefield epifluorescent or laser scanning microscopy can be used for high resolution and three-dimensional analysis. Fixation and preparation techniques were optimised to best preserve nuclear morphology and protein epitopes without the need for any antigen retrieval. Preparation of plant material involved short cross-linking fixation of meiotic tissues with paraformaldehyde (PFA) followed by enzyme digestion and slide-mounting. In order to avoid rapid sample degradation typical of shortly fixed plant materials, and to be able to perform IL later, slides were snap-frozen and stored at -80°C. Ultra-freezing produced a remarkable degree of ...Continue Reading

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Citations

Mar 29, 2020·PeerJ·Alexander P YoungRussell C Wyeth
Mar 5, 2021·Bio-protocol·Maria Ada PrusickiArp Schnittger
Jun 25, 2021·Current Protocols·Luis Munoz-ErazoKylie M Price
Jun 26, 2021·The Plant Journal : for Cell and Molecular Biology·Andrea Lenykó-ThegzeAdél Sepsi

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Methods Mentioned

BETA
electron microscopy
ImmunoFISH
PCR
Fluorescence
fluorescence microscopy
light scattering
confocal microscopy

Software Mentioned

Leica Application Suite Advanced Fluorescence
Huygens Essential

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