Serum donkey

Wash coverslips 3x with PBS and place them on drops of 5% normal donkey serum (NDS)/PBS for 10 min to block unspecific epitopes. 

Infiltration was done with 30% sucrose in PBS, sections were cut on a cryostat. Permeabilization was done with 0.3% Triton X-100 in PBS containing 5% donkey serum, 10 mg/ml of BSA. 

Rinses with PBS contained 5% normal donkey serum, 10 mg/ml of BSA. 

Place the slides in a humid chamber see Note 42) Expose the sections for 30 min at room temperature to PBS-TX-NS (using donkey serum) Dram off the solution, but do not rinse in buffer before the next step... 

Normal donkey serum (NDS, e.g.. Sigma Chemicals) or any other normal nonimmune serum. 

Coronal cryostat sections (10 pm thick) are cut after cryoprotec-tion and processed for indirect IMF. Sections are first incubated for 60 min with 10 % NGS or 10 % normal donkey serum in PBS to block nonspecific binding. They are then incubated with antibodies diluted according to manufacturers recommendations 1 h with primary antibody and then 1 h with secondary antibody diluted 1 100. Double IMF is performed according to standard protocols [48]. Mounting media contain DAPI to stain nuclei. Negative controls are performed each time immunostaining was done and consisted of preincubation with PBS, substitution of nonimmune isotype-matched control antibodies for the primary antibody, and/or omission of the primary antibody. Further details can be found in [48]. 

Blocking is made with 10 % donkey serum in PBS +0.2 % Triton X-100, 10 min. There is no washing after blocking. 

The sections are incubated with primary antibodies in blocking buffer at 37 °C or 4 °C overnight. They are washed in PBS (3x 5 min) before incubation with the secondary antibody during 45 min in 1 % donkey serum in PBS without Triton X-100. After washing in PBS (3x 5 min), sections can be mounted with a medium containing DAPI to visualize nuclei. 

For confocal microscopy, cerebellar sections are prepared from mice injected with DA-594 or lentivirus expressing GFP. TPBS is used as diluent and as the washing buffer. Free-floating DA-594-labeled sections are incubated with 10 % normal donkey serum in TPBS for 30 min, then with a mixture of VGluT2 and calbindin antibodies overnight (1 pg/mL), and subsequently with a mixture... 

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