Kaiser s glycerol gelatin

Fluorescence immunohistochemistry was performed as previously described (Hack et al., 2001; Junghans et al., 2008). For cryostat sections non-specific binding sites were blocked for 1 h at RT (10% normal goat serum (NGS), 0.5% Triton X-100 in PBS). The tissue was subsequently incubated overnight at 4°C with the primary antibodies (3% NGS, 0.5% Triton X-100 in PBS). After several washes in PBS sections were incubated for 1 h with fluorochrome-coupled secondary antibodies (3% NGS in PBS), washed in PBS and mounted (Kaisers glycerol gelatin, Merck).
Immunohistochemistry on paraffin sections including antigen retrieval was performed as previously described (Junghans, 2008). Peanut agglutinin (PNA) labeling of cone pedicles was performed as described previously [46 (link)].
Fluorescence imaging was done using an Axioscop2 and Axiovert 200M with ApoTome function (Zeiss, Germany) and images processed by using Zeiss AxioVision software.

To investigate the effects of TBMS1 on actin skeleton organization of eEND2 cells, we stained F-actin with phalloidin. For this purpose, eEND2 cells (1 × 10⁴ per well) seeded on glass coverslips in 24-well plates were exposed to vehicle or 10μM TBMS1. After 24h, the cells were fixed in 4% formalin for 10min at room temperature, washed twice in PBS and permeabilized for 10min with 0.2% Triton X-100, and then blocked in 1% bovine serum albumin (BSA) for 30min. Cells were then stained with Alexa Fluor 568-conjugated phalloidin (Invitrogen, Darmstadt, Germany) and Hoechst 33342 (Sigma-Aldrich) at room temperature for 30min and 10min, respectively. After two washes in PBS, slides were mounted in Kaiser's glycerol gelatin (Merck, Darmstadt, Germany) for fluorescence microscopy (BZ-8000; Keyence). The number of cells with stress fibers was assessed as a percentage of the total cell number. The analyses were performed with 3 repeats in 3 independent experiments.

Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X (Sigma). After washing three times each with PBS, the cells were blocked with 1% bovine serum albumin in PBS. Subsequently, the cells were incubated overnight at 4 °C with the primary antibodies. The cells were washed three times with PBS and incubated with Alexa Flour 488-labeled anti-rabbit IgG (Life Technologies, Carlsbad, CA). Nuclei were visualized by staining for 5 min with 1 μg/mL DAPI. After washing, the preparations were mounted using Kaiser’s Glycerol gelatin (Merck, Darmstadt, Germany). The fluorescence intensity of the preparations was detected using a confocal microscopy (Zeiss). Fluorescence intensity in cells per sections was measured with microscope image-analysis software (ZEN blue software) by a single investigator who was blind to sample identity. In each of four replicate experiments, 20 images were recorded, and the fluorescent cells in each image were counted (every image contained approximately 20 cells). Finally, the cell concentration was calculated from the average number of cells per image.

RNase and DNase enzymes were used from Invitrogen by Thermo Fisher Scientific RecoverAll Kit which is used for RNA purification. Proteinase was used from Cobas DNA Sample Preparation Kit, typically used for DNA extraction from FFPE tissue sections. Four serial sections were cut, and deparaffinized. Three of the sections were subsequently treated with 30 µl of each enzyme solutions, coverslipped with mounting glass and sealed with gum glue to ensure that the mixture did not evaporate. Each enzyme solution mix was prepared according to the manuals from the kits: proteinase solution mix—180 µl tissue lysis buffer, 70 µl proteinase K, incubation at 56 °C temperature for 1 h; DNase solution mix—4 µl DNase, 6 µl 10xbuffer, 50 µl water, incubation for 30 min. at room temperature; RNase solution mix—10 µl RNase, 50 µl water, incubation for 30 min. at room temperature. After incubation the gum glue was removed and sections were washed with distilled water, stained with Hoechst solution (500 µg/ml) for fluorescent nuclei visualization and coverslipped with aqueous mounting media (Kaiser’s glycerol gelatin, Merck). The same region from all four serial sections was imaged by PPM microscopy to compare the three enzymatically treated sections with the fourth treated with deparaffinization only.

For staining of murine PFA-fixed (ON at 4% PFA) LNs sections, tissues were dehydrated stepwise with 30%, 50%, 70%, 80%, 90%, and pure ethanol solution for 1 hour, respectively. After subsequent incubation in toluol, dehydrated LNs were embedded in paraffin and sectioned into 6 μm thick slices (HM355S Microtom, Microm).
Murine sections and human MTAs were heat treated at 60°C for 60 min. Prior to the staining procedure, paraffin was removed in xylol and acetone, and sections were rehydrated and treated with 10 mM citrate buffer for 5 min in a pressure cooker for antigen retrieval. All sections were blocked with avidin/biotin blocking solution (DAKO), incubated with the primary antibody overnight at 4°C and subsequently incubated with alkaline phosphatase (AP) or horse radish peroxidase (HRP) -conjugated secondary antibodies for 1 hour at RT. Fuchsin or 3-amino-9-ethylcarbazole (ACE) staining of sections was performed with the Fuchsin Substrate-Chromogen System or ACE substate (both DAKO) according to the manufacturer’s instructions. All sections were counterstained with Hematoxylin (ThermoFisher) and mounted with Kaiser’s glycerol gelatin (Merck Millipore). Bright field microscopy was performed using an Axio Imager 2 microscope and Axio Vision 4.8.2 Software (both Carl Zeiss).