Alexa 647 fluorophore

IHC of skin sections was performed with free-floating sections. Light antigen retrieval was performed in 0.1 M PB pH 7.3, 0.2% hydrogen peroxide, and 0.05% Triton X-100 for 25 min at room temperature (RT). Sections were then washed two times in PBS and incubated in blocking solution of Tris-buffered saline (TBS) with 0.05% Tween 20, 2% bovine serum albumin (BSA), and 2% normal donkey serum (NDS) for 1 h before applying a primary antibody overnight at 4°C. The following primary antibody (diluted in the blocking solution) was used: rabbit anti-PGP9.5 (1:500, ab108986, Abcam). For detection of the primary antibody, a secondary antibody raised in donkey and conjugated with Alexa-647 fluorophore was used (1:500, Molecular Probes, Thermo Fisher Scientific) for 1 h at RT. DAPI staining (1:2,000, Molecular Probes) was performed as the same time as the secondary antibody. Sections were then washed two times with PBS then placed on superfrost glass slides. One drop of Immu-Mount is added over the tissue sections, and a cover glass is placed over the slide.

Images of yeast cells and CW treated in different ways were obtained with fluorescent confocal scanning microscope Leica TCS SP2 AOBS (Leica, Rostock, Germany). Images of material from protein extracts (T and G pools) were obtained with fluorescent confocal scanning microscope Carl Zeiss Axiovert 200M LSM 510 META (Zeiss, Moscow, Russia).
Yeast cells, CW, and protein extracts (T and G pools) were fixed on glass slides with 3.7% paraformaldehyde for 20 min at room temperature incubated in conditions preventing desiccation. Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the Section 4.9) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany). Antibodies to Gas1 were kindly provided by Dr. M.O. Agaphonov (Federal Research Center “Fundamentals of Biotechnology”, Russian Academy of Sciences, Moscow, Russia).
Cells, CW, and protein extracts of bgl2Δ strain were used as the antibody controls to cells and CW of wt strain. Untreated with EDC CW of wt strain were used as the control to EDC-crosslinked CW of wt strain.