Goat anti rabbit igg af 647

Skin cell suspensions were obtained by Liberase digestion as mentioned above. Cell suspensions were diluted in RPMI and deposited in microplates containing poly-L-lysine-coated coverslips (Corning). Cells were incubated overnight at 4°C and fixed with 4% paraformaldehyde. Cells were then incubated with rat anti-mouse MHC-II (clone 2G9, BD Bioscience) and rabbit anti-clathrin heavy chain (Abcam), followed by AF647 goat anti-rabbit IgG (Invitrogen) and AF-555 goat anti-rat IgG (Invitrogen). Finally, coverslips were mounted on microscope slides using ProLong Gold with DAPI (Life Technologies). Cells were visualized with a LMS 700 confocal microscope (Zeiss) and pictures were edited using Zen software (Zeiss).

After infection, mice were euthanized, and the brain was removed after perfusion as described above. The brain was immediately frozen in OCT compound and cut on a cryostat microtome at a thickness of 5-μm sections onto coated glass slides. Tissue sections were fixed in ice cold acetone for 10 minutes. Sections were then incubated with 3% goat serum in PBS, followed by incubation with rabbit-anti-mouse collagen IV (Invitrogen), rat-anti-mouse F4/80 (eBioscience), and a mouse mAb specific for cryptococcal polysaccharide (E1, a gift from Françoise Dromer, Institut Pasteur, Paris) at 4°C overnight in a humidified chamber. After 3 washes, sections were incubated for 30 minutes with AF647 goat anti-rabbit IgG (Invitrogen) to delineate brain micro-vasculature, and with AF488 goat anti-rat IgG (Invitrogen) to identify monocytes, and AF555 goat anti-mouse IgG (Invitrogen) to stain the yeast cells in the brain microvasculature. The sections were rinsed and mounted with fluorescence anti-fading medium (KPL).

To check the injected area of AAVs, direct fluorescence of RFP was observed and microphotographs were taken with a fluorescence microscope (Keyence BZ-X800, BZ-X710; Osaka, Japan). Immunofluorescent staining against tyrosine hydroxylase (TH) was conducted to detect dopaminergic neurons and to outline the VTA and SNc. The anti-TH IHC method is described below. (1) To verify anti-TH immunoreactivity of the infected neurons in the midbrain and (2) to investigate anti-TH immunoreactivity of their axons at target areas, we applied IHC against the RFP to enhance the relatively weak signal in addition to the anti-TH IHC. Sections were incubated with monoclonal mouse anti-RFP (1:500, MBL, Woburn, MA USA; M155–3) and polyclonal rabbit anti-TH (1:500, Merck Millipore; Burlington, MA USA: AB152) in 5% NGS in PBS/T after blocking incubation. Subsequently, sections were visualized with AF555 Goat anti-mouse IgG (1:200, Abcam; Cambridge, UK: ab150117) for RFP, and AF647 goat anti-rabbit IgG (1: 200, Invitrogen; Carlsbad, CA USA: A-11012) or AF488 goat anti-rabbit IgG (1:200, Invitrogen; A-11008) for TH, respectively.

The sectioned samples were washed three times in DPBS, permeabilized with 0.5% Triton X-100 (Sigma) in DPBS for 15 mins, and blocked for 1 h with 1% bovine serum albumin (BSA, Sigma), 10% goat serum (Invitrogen), 0.3 M glycine (Fisher), and 0.1% Triton X-100 in DPBS. The samples were incubated overnight with the primary antibody [TRPV4 (Abcam, ab39260), RUNX2 (Abcam, ab76956), YAP (Santa Cruz, sc-101199), CD105 (R&D Systems, MAB1320), all used at 1:200 dilution]. After washing three times with 0.1% Triton X-100 in DPBS, DAPI and phalloidin (Invitrogen) were incubated for 1 h to stain the nucleus and F-actin along with secondary antibody, such as goat anti-Rabbit IgG AF647 (Invitrogen, 1:500 dilution) and goat anti-mouse IgG AF 555 (Invitrogen, 1:500 dilution).