Mowiol dabco

Drosophila S2 cells grown on cover slips, dissected ring glands and imaginal discs were processed as described [65] (link) and stained overnight at 4°C (tissues) or 2 h at room temperature (S2 cells) with the following antibodies: rat anti-Ecd^Nterm (1∶500, this study), guinea pig anti-Spok (1∶1000) and rabbit anti-Phm (1∶300) [33] (link), rabbit anti-cleaved Caspase-3 (1∶500, ASP175, Cell Signaling, #9661), rabbit anti-pH3 (1∶100, Cell Signaling, #9701), mouse anti-Flag M2 (1∶500, Sigma Aldrich), rabbit anti-c-Myc (1∶500, sc-789, Santa Cruz), and mouse anti-Lamin (1∶500, ADL67.10), mouse anti-EcR (1∶200, DDA2.7), and mouse anti-Fasciclin III (1∶300); the latter three from the Developmental Studies Hybridoma Bank (DSHB, Iowa). After washing, samples were incubated with corresponding secondary antibodies coupled to Cy3 or Cy5 (Jackson ImmunoResearch), counterstained with DAPI (0.5 µg/ml, Invitrogen) to visualize nuclei, and mounted in Dabco:Mowiol (Sigma-Aldrich).

EADs, wing discs and PGs dissected from third instar Drosophila larvae (7 days AEL) and Drosophila S2 cells were fixed for 25 min with 4% paraformaldehyde in PBS containing 0.1% Triton X-100 (PBS-T) and washed 3 times with PBS-T. After blocking in 0.3% BSA in PBS-T samples were incubated overnight at 4°C with the following primary antibodies at the indicated dilutions: guinea pig anti-Spok (1:1000, Ono et al., 2006 (link)), rabbit anti-Dcp-1 (1:500, Cell Signaling Technology) (RRID:AB_2721060), rat anti-Elav (1:500, DSHB) (RRID: AB_528217), mouse anti-p120 (1:300, DSHB) (RRID: AB_2088073), rabbit anti-GFP (1:300, Thermo Scientific) (RRID:AB_2536526), rabbit-anti-dPrp8-CTD (1:500, this study, Eurogentec), mouse anti-Flag M2 (1:500, Sigma-Aldrich) (RRID: AB_262044). After washing, the samples were incubated with the corresponding Alexa Fluor 488- or CY5-conjugated secondary antibodies (Thermo Scientific or Jackson ImmunoResearch) for 2 h at room temperature and counterstained with DAPI (1 µg/ml, Carl Roth GmbH) to visualize nuclei. Tissues were mounted on glass slides in Dabco-Mowiol (Sigma-Aldrich).

MDCK cells were seeded on glass slides and cultivated to confluence as described above. The cells were treated with 250 μM chitosan nanocapsules in supplement-free medium for 2 or 24 h. Prior to staining, the cells were fixed at 4 °C for 30 min in HEPES-buffered Ringer’s solution (10 mM HEPES, 5 mM glucose, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM KCl, 140 mM NaCl) containing 4% paraformaldehyde. The cells were then permeabilized with incubation buffer (HEPES-buffered Ringer’s solution, supplemented with 0.3% Triton X-100 and 0.1% bovine serum albumin) and nonspecific staining was blocked with incubation buffer supplemented with 2% bovine serum albumin. Chitosan nanocapsules were stained with 100 μg/ml CAP-sfGFP and cell surfaces were counterstained with 2.5 μg/ml Texas Red conjugated to WGA (Life Technologies GmbH, Darmstadt, Germany). Nuclei were stained with 500 ng/ml 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Sigma-Aldrich GmbH, Steinheim, Germany). Coverslips were mounted in DABCO-Mowiol (Sigma-Aldrich GmbH, Steinheim, Germany) and analyzed by SIFM using an inverted fluorescence microscope (AxioObserver.Z1, Zeiss, Jena, Germany) equipped with a structured illumination module (ApoTome, Jena, Germany). Three-dimensional rendered images of optical sections were calculated with ImageJ50 (link).

For quantification of apoptosis after MCAO, terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling (TUNEL) was performed on cryosections using the in-situ-cell death detection kit (Roche). Sections were air-dried for 10 min at RT and postfixed in 4% PFA for 30 min. Then, sections were washed twice in PBS for 5 min and subsequently permeabilized in 0.1% sodium citrate, 0.1% TrtionX-100 in PBS. Sections were washed twice in PBS for 5 min and incubated with TUNEL reaction mix (50 μl per section, TUNEL-enzyme: labeling solution, 1:7) in a humid chamber for 2 h in the dark. After washing in PBS and DAPI-staining for 5 min at RT sections were washed in PBS and mounted with Mowiol/DABCO (Merck/Sigma-Aldrich). For combined TUNEL and fluorescence immunostaining, immunohistochemistry was performed as described above. To quantify the percentage of apoptotic cells fluorescence images were acquired at 10x magnification in three optical fields of the penumbra and analyzed using ImageJ (NIH). The number of DAPI and TUNEL positive cells was counted manually. Experimenters were blinded for the treatment group.

After the incubation, cells were washed two times with DPBS, fixed with 4% paraformaldehyde in PBS for 30 min, and permeabilized with 0.2% Triton X-100 for 15 min at room temperature. Lamin-B (goat polyclonal antibody, sc-6216, Santa Cruz, Heidelberg, Germany) and secondary antibody Alexa Fluor 546 (donkey anti-goat, A11056, Invitrogen, Darmstadt, Germany) were used to stain the lamina of the nucleus. Cells were mounted on glass slides with Mowiol/DABCO (Sigma Aldrich, Taufkirchen, Germany).