Alexa fluor 555 conjugated donkey anti rabbit igg

Tissue sections (40 μm) and cultured cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. The samples were then blocked with 10% donkey serum and probed with anti-CD31 (RD, AF3628), anti-Laminin (Abcam, ab11575), anti-VE-cadherin (Santa Cruz, sc-9989), anti-occludin (Proteintech, 66,378-1-Ig), anti-claudin 5 (Thermo, 35-2500), anti-NRF1 (CST, 46743S), anti-CAV-1 (CST, 3267), anti-ZO-1 (Proteintech, 21,773-1-AP) or anti-LC3 (CST, 12741S) antibodies. The binding of primary antibodies was visualized with either Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Thermo, A31572), Alexa Fluor 647-conjugated donkey anti-mouse IgG (Thermo, A32787), or Alexa Fluor 488-conjugated donkey anti-goat IgG (Abcam, ab150133). The samples were then counterstained with DAPI (Thermo) and imaged using a Leica SP8 confocal microscope.

Tissue sections (40 μm) and cultured cells were fixed with 4% paraformaldehyde. The samples were subsequently permeabilized with 0.5% Triton X-100. The samples were then blocked in 10% donkey serum, followed by incubation with the following antibodies: anti-MAP2 (Sigma-Aldrich, M4403), anti-Aβ1–16 (BioLegend, SIG-39300), anti-Iba1 (Abcam, ab5076), anti-TFEB (Proteintech, 13372-1-AP), anti-LC3A/B (Cell Signaling Technology, 12741S), anti-LAMP1 (Abcam, ab24170), and anti-synaptophysin (Cell Signaling Technology, 9020). The binding of the primary antibodies was visualized using Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific, A31572), Alexa Fluor 488-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific, A21202), or Alexa Fluor 647-conjugated donkey anti-goat IgG (Abcam, ab150131). Finally, the samples were counterstained with DAPI, and confocal microscopy (Leica Thunder or Leica SP8 confocal microscope) was used to capture the fluorescence images.

After 24 h treatment, the rats were anesthetized and perfused with PBS and 4% paraformaldehyde subsequently. Then, the brains were harvested, fixed in 4% paraformaldehyde at 4°C overnight, dehydrated in graded sucrose, and cut into 10 μm‐thick slices. Blocked in 5% normal donkey serum (Abcam; cat. no. ab7475) at room temperature for 1 h, primary antibodies were then added to incubate the sections overnight at 4°C: CD31 (1:100, Abcam; cat. no. ab24590), Pink1 (1:100, Abcam; cat. no. ab23707), caspase‐1 (1:100, Abcam; cat. no. ab1872), caspase‐3 (1:100, Abcam; cat. no. ab179517), IL‐1β (1:100, Cell Signaling Technology; cat. no. 12703), IL‐1R1 (1:100, Santa Cruz Biotechnology; cat. no. sc‐689), Iba1 (1: 100, Abcam; cat. no. ab15690), TNF‐α (1:100, Abcam; cat. no. ab205587), and iNOS (1:100, Abcam; cat. no. ab15323). Washed with PBS and the second antibodies were added into the slices to incubate for 1 h at room temperature. The secondary antibodies include Alexa Fluor^® 488‐conjugated goat anti‐mouse IgG (1:200, Thermo Fisher Scientific; A‐28175), Alexa Fluor^® 555‐conjugated donkey anti‐rabbit IgG (1:200, Thermo Fisher Scientific; A‐31572). Then, sealed with fluorescent mounting medium (Sigma, St. Louis; cat. no. F6057), the slices were finally observed under the fluorescence microscope.

We used the antibodies in parentheses to the following antigens: ATG9A (Abcam, catalog #108338), FLAG M2 (Sigma, F1804), PAK3 (Abnova, PAB2300), SPTLC2 (Abcam, ab23696), GFP-HRP (MACS, 130091833), actin-HRP (Sigma, A3854), beclin 1 (Cell Signaling, 3738), ATG7 (Cell Signaling, 8558), ATG5 (Cell Signaling, 12994), ATG12 (Cell Signaling, 4180), ATG16 (Cell Signaling, 8089), LC3 (Cell Signaling, 3868), LAMP-1 (Cell Signaling, 9091), SNAP29 (Abcam, 138500), gp41 (NIH AIDS Reagent Program, 2F5), TGN46 (Bio-Rad, AHP500G), anti-HIV immunoglobulin (NIH AIDS Reagent Program, HIV-Ig), Nef (NIH AIDS Reagent Program, 2949), α-tubulin (Sigma, T5168), VSV-G (Sigma, V5507), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen, A21206), Alexa Fluor 488- conjugated donkey anti-mouse IgG (Invitrogen, A21202), Alexa Fluor 555-conjugated donkey antirabbit IgG (Invitrogen, A31572), Alexa Fluor 555-conjugated donkey anti-mouse IgG (Invitrogen, A31570), Alexa Fluor 555-conjugated donkey anti-sheep IgG (Invitrogen, A21436), HRP-conjugated donkey anti-rabbit IgG (GE Healthcare, NA934V), and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, NXA931).

Immunofluorescence was performed to determine the expression of proteins related to CAFs markers. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, then blocked with 5% bovine serum albumin, and incubated with goat anti-rabbit FAP (1:100, SAB, United States) and goat anti-mouse α-SMA antibody (1:150, BioWorld, United States) at 4°C overnight. The MSCs were then washed and incubated with Alexa Fluor 555 conjugated donkey anti-rabbit IgG or FITC conjugated goat anti-Rabbit IgG (Invitrogen, United States) for 1 h. The nuclei were stained with DAPI (1:200, Sigma-Aldrich) and images were acquired using a fluorescent microscope (Nikon, Japan). The experiments were repeated in triplicate for each group.