Superfrost micro slide glass aps coated

Free-floating immunostaining was carried out as follows. Tissue sections were washed twice for 10 min in 0.3% Triton X-100 in PBS (PBS-X) and reacted for overnight to 3 days with primary Abs in PBS-X containing 0.12% λ-carrageenan (035-09693; Sigma-Aldrich) and 1% normal donkey serum (S30-100ML, Merck Millipore) (PBS-XCD). After washing twice for 10 min in PBS-X, the sections were then reacted for 2 − 4 h with secondary Abs in PBS-XCD. The sections were washed twice for 10 min in PBS-X. Some of the sections were counterstained with NeuroTrace 435/455 Blue or NeuroTrace 530/615 Red Fluorescent Nissl Stain (1:100, N21479 or N21482, Thermo Fisher Scientific) in PBS-X. The sections were mounted onto glass slides (Superfrost micro slide glass APS-coated, Matsunami Glass) and coverslipped with 50% glycerol, 2.5% 1,4-diazabicyclo[2.2.2]octane, and 0.02% NaN₃ in PBS (pH 7.4). Endogenous peroxidase activity was quenched with 1% H₂O₂ in PBS for 30 min prior to immunostaining. For multiplex FT-GO IFs, the Ab-POD conjugate was inactivated with 2% NaN₃ in PBS for 4 h after each round of FT deposition. All incubations were performed at 20–25 ºC. Primary Abs (pAbs) and secondary Abs (sAbs) used are listed in Supplementary Table S1 and S2 online.

Bright-field images of tissue sections were obtained with a light microscope (BX-51, Olympus) equipped with dry objectives (10× UPlanApo, NA = 0.40, WD = 3.1 mm, Olympus; 40× UPlanApo, NA = 0.85, WD = 0.2 mm, Olympus) and a CCD camera (DP72 or DP74, Olympus). DAB-Ni²⁺-labeled sections were mounted onto glass slides (Superfrost micro slide glass APS-coated, Matsunami Glass) and coverslipped with 50% glycerol, 2.5% 1,4-diazabicyclo[2.2.2]octane (DABCO) (049-25712, Wako Pure Chemical Industries), and 0.02% sodium azide (31233-55, Nacalai Tesque) in PBS.