Pbs tx

Brains were fixed with 4% (w/v) paraformaldehyde (PFA, Roth, Karlsruhe, Germany) solution and 0.4% (w/v) Lucifer yellow for enhanced background (Lucifer Yellow CH dilithium salt, Sigma-Aldrich, München, Germany) in phosphate buffered saline [PBS; NaCl (37 mM), KCl (2.7 mM), Na₂HPo₄ (8 mM), KH₂Po₄ (1.4 mM), adjusted to pH 6.7 using diluted HCL] or a mixture of 1.3% (w/v) PFA and 0.7% (v/v) glutaraldehyde (GA, Sigma-Aldrich, München, Germany) in PBS for at least 4 h. Subsequently, the brains were washed three times in PBS for 10 min each, dehydrated in ascending ethanolic solution series [50, 70, 90, 99, and 100% (v/v), each for 10 min], cleared in methylsalicylate (Roth, Karlsruhe, Germany) for 10 min, and mounted on a special object slide (a metal plate of 0.5 mm thickness with a central hole and cover slips on both sides) in methylsalicylate. Additionally, brains stained with Neurobiotin were permeabilized for 2 h in PBS containing 0.5 or 1% (v/v) Triton X (PBS-TX, Sigma-Aldrich, München, Germany) and incubated overnight at 4°C in 0.05% (w/v) indocarbocyanine (Cy3) or indodicarbocyanine (Cy5) coupled streptavidin (Dianova, Hamburg, Germany) 1:100 in PBS-TX containing 0.1% (w/v) sodium chloride (NaCl) for enhancement. The following day, the brains were washed at room temperature several times (for at least 2.5 h) in PBS, dehydrated and mounted as described above.

At adulthood, brain sections were washed in phosphate buffer saline (PBS) and then incubated for 1 h in 10% normal donkey serum (NDS; Abcys, Paris, France) in PBS with Triton X-100 (PBS-Tx; Sigma-Aldrich, San Luis, MO, USA). Afterwards, they were incubated for 48 h at 4°C with the primary reagents cocktail, diluted in PBS-Tx, and 5% NDS. The primary reagents cocktail consisted of guinea pig anti-PV antibody (1:9,500, Synaptic Systems, Gottingen, Germany), and Wisteria floribunda agglutinin (WFA; 1:710, Sigma-Aldrich, San Luis, MO, USA) for the detection of PNN (Härtig et al., 1992 (link)). After washing, sections were incubated for 2 h at room temperature with matching fluorescently labeled secondary reagents also diluted in PBS-Tx: A488-conjugated avidin (1:400, Invitrogen, Waltham, MA, USA) and CF555 donkey anti-guinea pig secondary antibody (1:400, Biotium, Fremont, CA, USA). Finally, sections were washed in PB 0.1 M, mounted on slides, and coverslipped using a fluorescence mounting medium (Dako North America Inc., Santa Clara, CA, USA).

Immunohistochemistry was carried out using the approach previously reported by Diene et al. [24 (link)]. Following a 10-minute incubation with 3% H₂O₂, sections were blocked for 15 minutes at room temperature with 2% bovine serum albumin (BSA) in PBS containing 0.4% Triton X-100 (PBSTx, Sigma Chemical Co., USA), and then incubated for 48 hours at 4°C with polyclonal glial fibrillary acidic protein (GFAP) antiserum raised in rabbit (Z0334 Dako, 1:500, UK). After numerous washes with PBS-Tx, the sections were incubated at room temperature for 2 hours with biotin-conjugated goat anti-rabbit IgG secondary antibody (Amersham, 1:50, UK). After washing again, sections were incubated with horse radish peroxidase (HRP)-conjugated streptavidin at 37°C for 15 min. At the end of incubation, sections were stained by 3,3-diaminobenzidine (DAB, K3467, Dako) at room temperature for 15 min in the dark. All sections were examined under a light microscope (Olympus IX70, Olympus Optical Co. Ltd, Japan). Image J software was used to perform semi-quantitative densitometric analysis on the intensity of GFAP immunoreactivity.