Rabbit anti-calretinin

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Publication protocol

Slices were blocked in carrier solution (10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA), 2% Triton X-100 (Sigma-Aldrich, Buchs, Switzerland), and 0.04% NaN3 in 0.1 M PBS) overnight at room temperature on either a shaker (IKA-VIBRAX-VXR, IKA, Staufen, Germany) or a rotation incubator (CMV-ROM, Fröbel Labortechnik, Lindau, Germany). Slices were then incubated with primary antibodies (Table 1) in carrier solution for 3 days at room temperature on the shaker. Afterwards, they were briefly rinsed three times with 0.1 M PBS, then washed three times with PBS for 30 min, and subsequently incubated with secondary antibodies (Table 1) in carrier solution for 1 day at room temperature on the shaker or rotation incubator, respectively. After further rinsing and washing (three times each), slices were cleared in a sequence of sucrose solutions (15%, 30%, 45% and 60% of sucrose in 0.1 M PBS/2% Triton X-100) with 4-h periods per sucrose concentration. Negative controls were separately performed for each secondary antibody. No false-positive candidate cell bodies were detected.

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