Dapi containing fluoroshield

Sections were deparaffinized and rehydrated. Heat-induced antigen retrieval was carried out in 0.1 M sodium citrate (pH 6) buffer. Tissue sections were then blocked in 1% BSA, 0.1% Triton-X, and 1% serum. Sections were then treated with primary antibodies overnight at 4°C. For BrdU immunofluorescence, sections were additionally treated for 30 min with 2 M HCl at 40°C before incubation with primary antibody. After secondary antibodies, slides were mounted with Fluoroshield containing DAPI (Abcam). Sections were imaged with a Leica TCS SP5 confocal microscope. To ensure true and specific protein expression, positive and negative (without primary antibodies) procedural controls were maintained. Images were processed using Fiji (Schindelin et al., 2012 (link)). See supplemental experimental methods for antibody details.
Tartrate-resistant acid phosphatase (TRAP) staining was carried out as described in Grigoriadis et al. (1994) (link).

Brain slices were post-fixed with 10% formalin, washed three times with PBS, incubated for 30 min with 0.3% Triton, and then blocked with 1% goat serum for 1 h to avoid binding of non-specific antibodies. Sections were then incubated at 4°C overnight with the following primary antibodies: anti-NKCC1 (Millipore, PA5-118800, Boston, MA, USA, 1:200), anti-NeuN (Sigma-Aldrich, St. Louis, MO, USA, MAB377, 1:200). Secondary antibodies conjugated to Alexa Fluor 488 or 594 (1:1,000; Invitrogen) were applied for 1.5 h at room temperature. Slides were mounted with Fluoroshield containing DAPI (Ab104139; Abcam) and observed with a confocal microscope (LSM710; Zeiss, Oberkochen, Germany).

Slides were double‐stained for Marginal zone B and B1 cell‐specific protein (MZB1; HPA043745, Atlas Antibodies AB, diluted 1:2000) and Alpha defensin 1 (DEFA; HPA052517, Atlas Antibodies AB, diluted 1:600) and nuclei were visualized with DAPI. Deparaffinization, antigen retrieval, blocking, incubation with primary antibodies, secondary HRP polymer, and washing steps were performed in the same manner as for immunohistochemistry, described above, with the addition of two additional washing steps. For visualization, slides were incubated with Tyramide Signal Amplification (TSA) Substrate (TSA‐Plus, PerkinElmer, MA, USA) for 15 min. Each antibody was added in a separate staining cycle and visualized with different TSA fluorophores; Cyanine 3 (MZB1, yellow) and Cyanine 5 (DEFA1, red), diluted in amplification diluent (PerkinElmer). Between the cycles, slides were boiled in retrieval buffer at 90°C degrees for 20 min for elution of the first primary antibody. Finally, slides were mounted with Fluoroshield containing DAPI (Abcam, Cambridge, UK).