Alexa fluor 488 goat anti mouse secondary antibody

Cells were plated at a density of 2000 in 24-well chamber slides and treated with different concentrations of cisplatin for 24 hours. The cells were fixed with MeOH and permeabilized with Triton X-100. Then cells were blocked with 1%BSA in PBS for 2 hours, and incubated with primary antibody against γ-H₂AX (Biolegend, San Diego, CA, USA, 1:200 dilution) overnight. Finally, the cells were incubated with AlexaFluor-488 goat-anti-mouse secondary antibody (Jackson Immuno Research Laboratories, Inc., PA, USA, 1:200 dilution) and mounted with DAPI. γ-H₂AX foci were assessed with green fluroscence. At least three independent experiments were conducted.

Cell samples were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Tissue samples were fixed in 4% paraformaldehyde in PBS overnight at room temperature. The tissue samples were embedded, frozen, and sliced. Both the cell and tissue samples were then washed, permeabilized, and blocked. We used the following antibodies to investigate protein expression in these samples: anti-Piezo1 (Abcam, ab128245, 1:100), anti-Ki67 (CST, #9449, 1:400), anti-F-actin (Abcam, ab130935, 1:500), anti-α-SMA (CST, #48938, 1:200), Alexa Fluor 594 goat anti-rabbit secondary antibody (Jackson lab, 125369, 1:200), and Alexa Fluor 488 goat anti-mouse secondary antibody (Jackson lab, 133384, 1:200). Images were captured using a confocal microscope (Zeiss, Germany) or a Nikon Eclipse E800 microscope (Nikon, Melville, NY, USA).

For immunofluorescence-based detection of PrP^Sc aggregates in astrocytes, 1 × 10⁵ cells of prion-infected (22L, RML, and ME7) astrocytes were seeded in 12-well culture plate. Once 60–70% confluent, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by quenching of autofluorescence (with 50 mm NH₄Cl, 20 mm glycine) for 10 min at room temperature. The cells were permeablized with PBS containing 5% FBS and 0.3% Triton X-100 for 30 min followed by denaturation of PrP^C and epitope retrieval of PrP^Sc by incubation with 6 m guanidine HCl for 10 min at room temperature as described previously (44 (link), 45 (link)). The cells were incubated with anti-PrP mAb 4H11 (diluted at 1:100 in PBS containing 5% FBS and 0.3% Triton X-100) overnight at 4 °C. Alexa Fluor 488 goat anti-mouse secondary antibody (Jackson Immunoresearch) was used (at 1:200 in PBS containing 5% FBS and 0.3% Triton X-100) for 1 h at room temperature to visualize immunofluorescence. 4′,6′-Diamino-2-phenylindole (1:5000 in PBS) was used as a nuclear stain. Glial fibrillary acidic protein rabbit pAb was purchased from DAKO (Z0334). All images were captured under 63× oil lens at the same acquisition settings from a Zeiss LSM 700 confocal microscope.

Cell samples were fixed with 4% PFA for 15 min at RT. Tissues were fixed with 4% PFA overnight at RT. Tissue samples were embedded in paraffin and sliced into 5 mm sections. Samples were incubated with following antibodies: Piezo1 (ab128245, 1:100, Abcam), α-SMA (ab7817, 1:100, Abcam), CCL24 (22306-1-AP, 1:200, Proteintech), CCR3 (ab32512, 1:100, Abcam), Wnt2 (A5864, 1:200, Abclonal, Wuhan, China), Wnt11 (ab31962, 1:200, Abcam), β-catenin(#8480, 1:100, CST), Alexa Fluor 594 goat anti-rabbit secondary antibody (Jackson lab, 125369, 1:200) and Alexa Fluor 488 goat anti-mouse secondary antibody (Jackson lab, 133384, 1:200). Images were visualized using a Nikon Eclipse E800 microscope (Nikon, Melville, NY, USA).

Samples were fixed with 4% paraformaldehyde overnight, then embedded in paraffin. 5 mm sections were processed and stained with hematoxylin and eosin (H&E) and Masson’s Trichrome Stain Kit following protocol (Solarbio, Beijing, China).
For immunohistochemistry and immunofluorescence, 5 mm sections were incubated with primary antibodies against Piezo1 (ab128245, 1:1000, Abcam), α-SMA (ab7817, 1:200, Abcam), Collagen I (ab6308, 1:200, Abcam), GFP (ab290, 1:500, Abcam), Arg1 (16001-1-AP, 1:400, Proteintech), OAT (A6235, 1:100, ABclonal Technology), PYCR1 (13108-1-AP, 1:200, Proteintech), PRODH (22980-1-AP, 1:400, Proteintech) overnight at 4 °C. Sections were then incubated with Alexa Fluor 594 goat anti-rabbit secondary antibody (125369, 1:200, Jackson lab), and Alexa Fluor 488 goat anti-mouse secondary antibody (133384, 1:200, Jackson lab) for immunofluorescence or an HRP-conjugated goat anti-rabbit secondary antibody (138729, 1:500, Jackson lab) for immunohistochemistry. Images were captured using a Nikon Eclipse E800 microscope (Nikon, Melville, NY, USA).