9000s sliding microtome

Mice were deeply anaesthetized and trans-cardially perfused with 4% PFA. After removal of the spinal cord, spinal cord was post-fixed in 4% PFA overnight and subsequently incubated in 30% sucrose at 4 °C overnight. Thirty-five micrometers of thick free-floating sections were cut on a Leica 9000 s sliding microtome as described previously^{62 (link)} and collected in 0.1 M phosphate buffer (PB) pH 7.4. After incubation of the sections for 1 h with 4% normal goat or donkey serum and 0.3% Triton X-100 for blocking of nonspecific binding at room temperature, sections were incubated overnight at 4 °C with primary antibodies in blocking solution. After three times 10 min washing in 0.25% Triton X-100 in PB at room temperature, sections were incubated in with fluorescently labeled secondary antibodies, washed again and finally mounted with Mowiol/DABCO. The following primary antibodies were used: anti-NeuN (1:1000; Millipore, MAB377, clone A60), ChAT (1:1000; Millipore, MAB144P), anti-Ubiquitin (1:500; DAKO, Z0458), anti-Synaptophysin-1 (1:500; Synaptic Systems, 101 004). Alexa-647-, Alexa-488-, and Alexa-546-conjugated secondary antibodies were from Jackson Immuno-Research Laboratories. For visualization of F-actin Alexa Flour 532 conjugated Phalloidin (Invitrogen) was used.

Formalin-fixed livers were sectioned using a Leica 9000s sliding microtome (Wetzlar, Germany) into 40-μm-thick free-floating sections. After blocking with 4% normal goat serum and permeabilization with 0.5% Triton X-100 in 0.1 M phosphate buffer (PB), sections were incubated with appropriate primary antibodies overnight. Antibodies used for immunofluorescence were: rat monoclonal anti-CD68 (1:500, FA-11, AbD Serotec, Oxford, UK); rabbit polyclonal anti-myeloperoxidase (1:300, Millipore, Billerica, MA, USA); rat monoclonal anti-F4/80 (1:25 cell culture supernatant). The monoclonal antibody against mouse Lamp1 (clone 1D4B) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. Rat monoclonal anti-A6 (cell culture supernatant; 1:25) was a kind gift from Valentina Factor and described previously (Engelhardt et al., 1990 (link)). Cathepsin D antibody was described previously (Claussen et al., 1997 (link)). After washing with 0.25% Triton X-100 in PB and incubation with Alexa-Fluor-488 or -633 secondary antibodies (Molecular Probes, Eugene, OR, USA), sections were counterstained with DAPI and mounted with Mowiol/DABCO. Confocal laser scanning microscopy was performed on a Leica TCS SP2 microscope (Wetzlar, Germany).

4% Formaldehyde-fixed lungs were incubated in cryoprotectant solution (30% w/v sucrose in 0.1 M phosphate buffer, pH 7.4) over night. 35 μm thin sections were cut with a Leica 9000s sliding microtome (Leica, Wetzlar, Germany) and collected in cold 0.1 M phosphate buffer (PB). Free floating sections were stained by blocking in blocking solution (0.5% Triton-X 100, 4% normal goat serum in 0.1 M PB pH 7.4) for 1 hour at room temperature. Subsequently, sections were incubated in blocking solution containing the primary antibody at 4°C over night. After washing three times with wash solution (0.1 M PB pH 7.4 containing 0.25% Triton-X 100), sections were incubated for 2 hours in secondary antibody in solution, washed again three times in wash solution containing 4′,6-Diamidin-2-phenylindol (DAPI) and finally brought on glass slides and embedded in Mowiol/DABCO. Cells were imaged using a Zeiss confocal microscope equipoped with a 63x objective (Zeiss LSM980 AiryScan 2).