Computational protocol: Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB crystallin (HspB5) towards Hsp27 (HspB1)

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Protocol publication

[…] One and two-dimensional immunoblots were performed as already described , . They were probed with antibodies specific to the targeted proteins before being incubated with either goat anti-mouse or anti-rabbit immunoglobulin conjugated to horseradish peroxidase (Santa Cruz Biotechnology-Tebu, Le Perray en Yvelines, France) and subsequently revealed with ECL (Amersham Corp., Buckinghamshire, UK). Autoradiographs were recorded onto X-Omat LS films (Eastman Kodak Co, Rochester, USA). Films were scanned using a 4990 Epson film scanner and analyzed for quantification with ImageJ software™ (NIH, Bethesda). The duration of the exposure was calculated as to be in the linear response of the film. [...] HeLa cells (104/cm2) growing on glass cover slips were fixed for 10 min with freshly prepared 3.7% formaldehyde pH 7.0 in Phosphate buffered saline (PBS), before being permeabilized for 5 min in ice-cold acetone. F-actin was stained for 20 min with Alexa Fluor™ 488 Phalloidin (5U per ml of PBS) and vimentin was detected by incubating the coverslips for one hour with monoclonal anti-vimentin antibody (diluted 1/200 in PBS containing 0.1% bovine serum albumin IgG free). Other cover-slips were used to detect HspB1 and HspB5 using the corresponding anti-HspB1 monoclonal and anti-HspB5 polyclonal antibodies (diluted 1/100). After washing, HspB1, HspB5 and vimentin staining were revealed by incubating the cover-slips for one hour with FITC- and TRITC-conjugated goat-anti mouse or rabbit immunoglobulins (1/200 in TBS-Tween containing 0.1% IgG-free bovine serum albumin). Control experiments performed with non-immune sera or only the second antiserum confirmed that all detectable HspB1, HspB5 and vimentin fluorescences were specific. Hoechst 33258 staining was used to detect nuclei. The stained cells were then examined and photographed using in an LSM510 laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany) using a 63$ (numerical aperture, 1.4) Zeiss Plan Neo Fluorobjective. Illumination sources were 488 nm and 543 nm. To avoid cross talk between the different fluorochromes, the multitrack recording module was used, which allows a sequential acquisition of each channel. Individual as well as merge analysis were performed. Overlap and Pearson's coefficients were determined using the JACoP plug-in of ImageJ (NIH, Bethesda, USA). Computerized image analysis was performed using AxioVison LE software (Carl Zeiss MicroImaging GmbH, Germany). […]

Pipeline specifications

Software tools ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Cataract, Muscular Diseases