Alexa flour 594 conjugated goat anti rabbit igg

Vascular tissues were collected and fixed overnight in 4% paraformaldehyde (Solarbio, China), dehydrated, and embedded in paraffin. Sections were cut to a thickness of 4 μm, and an HE automatic staining instrument (HistoCore SPECTRA ST, Leica, Germany) was used to perform the HE staining. Images were taken using a digital slice scanner (KFBIO, China). The degree of stenosis and the neointima/media ratio were calculated as previously described (36 (link)).
For tissue immunofluorescence staining, paraffin sections were dewaxed in xylene and antigen-retrieved in citrate (Solarbio). Sections were blocked in QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime) for 1 h at room temperature and incubated with anti-α-SMA (1:200; Proteintech, Cat# 67735-1-Ig) and anti-PCNA (1:100; Proteintech, Cat# 60097-1-Ig) primary antibodies overnight at 4°C. Fluorescent secondary antibodies Alexa Flour 594-conjugated goat anti-rabbit IgG (1:200, Proteintech, Cat# SA00013-4) and Alexa Flour 488-conjugated goat anti-mouse (1:200, Proteintech, Cat# SA00013-1) were used to create fluorescent signals. Sections were observed with a laser confocal microscope (Nikon, AXR, Japan).

For cellular immunofluorescence, VSMCs were fixed with pre-cooled methanol for 5 min, blocked with 10% goat serum, and incubated with the following primary antibodies at 4 °C overnight: anti-SLC6A6 (1:50; Abcam, MA, USA) and anti-α-SMA (1:100; Proteintech, Wuhan, China). Paraffin sections were subjected to routine dewaxing and antigen retrieval. The sections were blocked in 10% goat serum at room temperature for 1 h and incubated with the following primary antibodies at 4 °C overnight: anti-SLC6A6 (1:100; Abcam, MA, USA), anti-α-SMA (1:200; Proteintech, Wuhan, China), anti-cyclin D1 (1:50; Abcam, MA, USA), and anti-PCNA (1:100; Proteintech, Wuhan, China). Immunohistochemical staining was performed with a DAB kit (ZSGB-BIO, Beijing, China) according to the instructions. Images were obtained using a digital slice scanner (KFBIO, Ningbo, China). For immunofluorescence staining, cells or vascular samples were incubated with the fluorescent secondary antibodies Alexa Flour 488-conjugated goat anti-mouse (1:200, Proteintech, Wuhan, China) and Alexa Flour 594-conjugated goat anti-rabbit IgG (1:200, Proteintech, Wuhan, China) for 1 h at room temperature. Nuclei were stained with DAPI (Solarbio, Beijing, China). Fluorescent images were obtained using a confocal laser microscope (Nikon, A1R, Tokyo, Japan) [32 (link)].

To visualize the expression of HMGB1 on mouse brain and BV2 cells, immunofluorescence staining was performed as described in our previous research.³³ In brief, after perfusion and fixation, the infarcted cerebral hemisphere (n = 3 per group) was prepared into paraffin sections. Brain sections (6 µm‐thickness) were sliced and blocked with 5% FBS at room temperature for 1 hour. BV2 cells were fixed in 4% paraformaldehyde for 15 minutes and washed twice with PBS. Then the cells were permeabilized by using 0.3% Triton‐100 in PBS at room temperature for 20 minutes. Thereafter, samples of brain sections and BV2 cells were incubated in rabbit anti‐HMGB1 antibody (1:100; Abcam) at 4°C overnight. After rinsed three times with PBS, samples were incubated with Alexa Flour 594 conjugated goat anti‐rabbit IgG (1:500; Proteintech) at room temperature for 1 hour. Cell nuclei were counter‐stained with DAPI (Beyotime) at room temperature for 3 minutes. All fluorescent images were captured and imaged by a fluorescence microscope (Carl Zeiss).