Glutathione s transferase

Cryopreserved liver, brain and cerebellum samples from were cut on a cryostat at 14 μm thin sections and placed on glass slides. Sections were brought to RT for 30 min, fixed in 4% paraformaldehyde for 20 min and washed with PBS. For Filipin staining tissues were incubated with 25 μg/mL Filipin (Sigma) over night at 4 °C protected from the light. After washing, slices were mounted with prolong antifade mountant (Dako). For GST-PFO staining, the sections were permeabilized with 0.2% Triton X-100 in blocking buffer (5% goat serum + 1% BSA in PBS) for 2 h in a dark-humid chamber. Then, slices were incubated 3 h with the probe GST-PFO (20 μg/ml) in 1% Goat Serum containing 0.05% Triton X-100 in PBS. After washing × 3 with PBS, samples were incubated O.N. at 4 °C with primary antibody Glutathione-STransferase (GST) (Santa Cruz). Secondary antibodies were diluted 1:200 in 1% Goat Serum containing 0.05% Triton X-100 in PBS and incubated for 90 min at RT with the mix of anti-mouse Alexa fluor-532 (for GST). After washing, slices were incubated 5 min in sudan black 0.1% in 70% EtOH to minimize autofluorescence and mounted with prolong antifade mountant (Dako). Images for all samples were taken with a Leica TCS SP5 laser scanning confocal system with a 633 oil immersion objective APO CS numerical aperture 1.4 equipped with a DMI6000 inverted microscope.

The AtFKBP16-1 antibody was prepared from mature protein of FKBP16-1 (72–207 aa), was amplified and then expressed into the pGEX4T-1 vector (Novagen, Darmstadt, Germany). The FKBP16-1::GST fusion protein was expressed by 0.2 mM isopropylthio-β-galactosidase for 2 h, and then the recombinant proteins were purified with glutathione-sepharose 4B resin (GE Healthcare, Uppsala, Sweden) according to the manufacture protocol. Polyclonal antisera were raised from own FKBP16-1 soluble protein in rabbit. Photosystem antibodies, PsaL (AS06 108), PsaF (AS06 104) and PsaA (AS06 172) for PSI; PsbA (AS11 1786) and PsbD (AS06 146) for PSII; PC (AS06 141) and cytochrome f (AS08 306) for electron transfer; and Lhcb1 (AS01 004) for LHC detection were purchased from Agrisera (Vännäs, Sweden). The glutathione-S-transferase (GST) and HA antibodies were used from Santa Cruz Biotech (Santa Cruz, CA) and Sigma (St Louis, MO), respectively.