Hc pl apo cs2 63x 1.4 oil objective

The samples were mounted on microscope slides, and images were captured using a Leica TCS SP8 microscope (Wetzlar, Germany) with the HC PL APO CS2 63x/1.4 oil objective at the Laboratory of Microscopic Imaging and Specialized Biological Techniques University of Lodz. The 488 nm laser was used to excite the fluorescence, and the emission was collected by a hybrid detector in the range of 505–550 nm. To visualize the cells, the PMT transmission channel was used. LAS X 2.0.2.15022 software (Leica Microsystems, Wetzlar, Germany) was used to analyze the data. All settings were held constant throughout the experiments. All signals obtained from confocal microscopy were validated with profile view image analysis, and the diagrams presenting intensity values were placed under each microphotograph. The mean fluorescence intensity (expressed in arbitrary units (AU)) was calculated for each of the samples. The calculations were performed for at least 40 different points randomly selected in compartments with receptor expression.

One day before transfection, HEK293 cells were plated into 0.001% poly L-lysine coated cover slips. Cells were transiently transfected with 0.5 μg of different HSPB5 mutants and either 1.5 μg of HSPB4-V5 or FRTTO for 48 h. Cells were washed twice with PBS, fixed with 3.7% formaldehyde in PBS for 15 min and permealized with 0.2% Triton X-100 in PBS for another 15 min. Cells were blocked with 100 mM glycine for 10 min then with 3% BSA in PBST for 30 min. Cells were incubated with primary antibodies: mouse anti myc (1:200 in PBS,MBL Japan) and rabbit anti V5 (1:200 in PBS, Invitrogen) overnight at 4 C°. Coverslips were washed with PBS-T (3x 5 min) and incubated with anti-rabbit Alexa fluor 488 (1:250 in PBST, Invitrogen) and anti mouse Alexa Fluor 594 (1:250 in PBST, Invitrogen) for 1 h. at room temperature. Coverslips were washed again with PBS-T and incubated with 0.2 μg/ml 4,6-diamidino-2-phenylindole (DAPI) for 10 min to stain the nuclei. Coverslips were washed with PBS and mounted with Citifluor medium (Citifluor Ltd, London, UK). Images were obtained with Leica TCS SP8 confocal laser scanning microscope with HC PL APO CS2 63x/1,4 oil objective.