Mouse anti cd68

Frozen tissue samples were sectioned from the aorta at the origins of the aortic valve leaflets (5-µm thickness) in a cryostat. Then each sample was fixed for 20 min in 4% paraformaldehyde solution in PBS followed by permeabilization with 0. 1% Triton X-100 in PBS for 20 min. After washing three times with PBS, sections were incubated with 10% goat serum in PBS for 1 h at room temperature to block nonspecific binding. Then the sections were incubated overnight at 4°C with primary antibody either rabbit anti-SRA1 (1:200) (abcam), mouse anti-ABCA1 (1:200) (abcam), or mouse anti-CD68 (1:200) (abcam) in 2% BSA in PBS. After washing three times with PBS, the sections were incubated for 1 h in 2% BSA in PBS containing a 1:500 dilution of the appropriate secondary antibody, either a goat anti-rabbit IgG conjugated with Chromeo™ 546 or goat anti-mouse IgG conjugated with Alexa Fluor^® 488 (abcam) or goat anti-mouse IgG conjugated with Chromeo™ 546 (abcam). The sections were then incubated with Hoechst for 5 min to stain the cell nuclei after washing three times with PBS. The sections were again washed three times with PBS and mounted with glycerol followed by observing on a ZEISS LSM 710 Laser scanning confocal microscope or common fluorescence microscope. The confocal images were analyzed with ZEN 2009 Light Edition software.

After blocked with BSA for 30 minutes, sections (5 μm) of abdominal arteries were incubated with rabbit anti‐COX‐2 (1:100, Abcam), mouse anti‐CD68 (1:100, Abcam) or mouse anti‐α‐SM actin (1:100, Sigma‐Aldrich) overnight at 4°C. After incubated with secondary antibodies, a drop of Prolong Gold anti‐fade reagent with DAPI (Vector Laboratories) was used to seal the coverslip. Images were acquired by laser scanning confocal microscopy (LSM 710, Zeiss).

Immunohistochemistry was performed on formalin-fixed and paraffin-embedded 4-μm sections of liver tissue. Sections were incubated overnight at 4°C with appropriate concentrations of primary antibodies, including rabbit anti-CD4, mouse anti-CD19, rabbit anti-CD38, mouse anti-CD68, and mouse anti-CXCR5 (Abcam, Cambridge, MA, USA) and goat anti-CXCL13 (R&D System, USA), and then incubated with the Dako Chemate Envision Kit (Dako, Glostrup, Denmark). The reaction was visualized by CheMate™ DAB plus chromogen (Dako, Denmark). The staining was captured by light microscopy using high-power microscopic fields (400×).

Cells were plated on glass coverslips in 24-well culture plates at a density of 50,000 cells per well. Coverslip cultures were fixed in 4% paraformaldehyde after 24 hours, permeabilized with 1% Triton X-100 in phosphate-buffered saline, and blocked with 0.2% Triton X-100 in 10% goat serum in phosphate-buffered saline. Samples with the following primary antibodies were incubated at 4°C overnight: for HSC staining, rabbit anti-α-SMA (1:100, Abcam), for Kupffer cell staining, mouse anti-CD68 (1:50; Abcam), and for cholangiocyte staining, rat anti-CK19 (1:250, Merck). On the next day, the following secondary antibodies were added for 2 hours in the dark at room temperature: Alexa Fluor 546–conjugated goat anti-rabbit (Thermo Fisher Scientific, diluted 1:500), Alexa Fluor 546–conjugated goat anti-mouse (Thermo Fisher Scientific, diluted 1:500), or Alexa Fluor 594–conjugated goat anti-rat (Thermo Fisher Scientific, diluted 1:500) + Hoechst (Invitrogen, diluted 1:2000) in 5% bovine serum albumin in phosphate-buffered saline. Slides were mounted with glass coverslips using 10 μL of mounting medium (ProLong Gold Antifade) and sealed with nail polish. A Leica DMC5400 fluorescence microscope (Leica Microsystems) with Leica LASX image acquisition software was used to visualize slides.

Rats were euthanized using pentobarbital (100 mg/kg, intraperitoneal) and perfused intravascularly with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). Brains were harvested and sectioned into 18-μm-thick slices with a cryostat after embedding. Immunohistochemical and immunofluorescence studies were performed as previously described [13 (link)]. The primary antibodies were rabbit anti-HO-1 (1:400 dilution; Abcam, Cambridge, USA), goat anti-Iba-1 (1:400 dilution; Abcam), mouse anti-CD68 (1:100 dilution; Abcam), mouse anti-rat CD163(1:100 dilution; AbD Serotec, Hercules, USA), polyclonal rabbit anti-alpha smooth muscle actin (1:200 dilution; Abcam). The secondary antibody in the immunofluorescence studies was Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen, Carlsbad, USA). Nuclear labeling was performed using fluoroshield™ with DAPI (F6057). Negative controls were performed without primary antibodies.