Cr7697

For detection of neural marker proteins, sections were rinsed 2 × 10 min and immersed in a blocking media of TBS containing 4% NDS, 2% BSA and 0.1% Triton X-100 for 1 h at RT. Sections were then incubated in primary antibody solution containing 1:10 mouse anti-RLN3 (as above) and either 1:2,500 rabbit anti-PV, 1:2,500 (PV27, RRID:AB_2631173, Swant), 1:5000 rabbit anti-CB28kD (CB38, RRID:AB_10000340, Swant), 1:1,250 rabbit anti-CR, (CR7697, RRID:AB_2619710, Swant), or (1:1000 guinea pig anti- Syn polyclonal, Cat. No. 101004, RRID:AB_1210382, Synaptic Systems, Göttingen, Germany) in TBS containing 2% NDS, 2% BSA and 0.2% Triton X100 for 48 h at 4°C. Sections were then rinsed (3 × TBS) and incubated in 1:200 FITC-labeled donkey antirabbit IgG (Cat No. 711-095-152, Jackson ImmunoResearch) and 1:200 Texas Red-labeled donkey anti-mouse IgG (Cat No. 715-075-150, Jackson ImmunoResearch); for quadruple labeling 1:200 Cy5labeled donkey anti-mouse IgG (Cat No. 715-175-020, Jackson ImmunoResearch) or 1:200 Cy5labeled donkey anti-goat IgG (Cat No. 705-175-003, Jackson ImmunoResearch) in TBS. Sections were then briefly rinsed in 0.01 M PBS and mounted on chrome-alum gelatin-coated slides, air-dried, dehydrated in graded ethanol and coverslipped with DPX.

Paraffin-fixed sections of 5 μm thickness were taken from three different blocks per human hippocampus. Per patient six sections two per each block were analyzed. Tissue sections were dewaxed with xylene and rehydrated with decreasing concentrations of ethanol. Unmasking was performed using 1 mM EDTA (pH 8) at 110°C in a pressure cooker for 5 min. Sections were afterward incubated with 3% H₂O₂ for 30 min, blocked for 1 h in TBS⁺ (containing 10% donkey serum) and subsequently Avidin-Biotin blocked (SP2001, Vector labs) followed by incubation with calretinin antibody (1:2000, CR7697, Swant) overnight at 4°C in TBS⁺ (containing 5% donkey serum).
To determine the number of labeled cells, the peroxidase method (Vectastain Elite ABC kit, Vector laboratories) was used with biotinylated donkey anti-rabbit antibody (1:500, Dianova) and diaminobenzidine (DAB, Sigma) as a chromogen. Sections were finally haematoxylin counter stained (Richard-Allan Scientific Hematoxylin, Thermo Scientific), sequentially dehydrated and cover-slipped with Entellan (Sigma-Aldrich).