Normal donkey serum

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

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

Rinses with PBS contained 5% normal donkey serum, 10 mg/ml of BSA. Donkey anti-goat IgG labeled with 546 fluorophore 1 1000 was used. 

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]. 

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... 

Normal serum may be rabbit, rat, goat, or donkey to match the choice of primary and secondary antisera. Available from DAKO, Carpinteria, CA... 

Always use normal serum from the species in which the secondary antibodies were produced. This is to block unspecific binding of the secondary antibodies. Thus, goat serum is appropriate if your secondary antibodies are produced in goat, donkey normal serum if the secondary antibodies were produced in donkeys, etc. 

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