Alexa fluor 488 affinipure goat anti rabbit igg

For immunofluorescence assay of tissue sections, deparaffinizated sections described above were washed in phosphate-buffer saline (PBS) for 5 min. These sections were boiled in sodium citrate buffer for antigen retrieval for 15 min, and blocked in 5% BSA for 45 min. For immunofluorescence of spermatozoa, slides were treated with 3-Aminopropyl (APES, Sigma, 919-30-2) in advance. 50 μl of sperms at 1 × 10⁶/ml was added onto the APES treated slides. Slides were fixed in 4% PFA, and blocked in 5% BSA supplemented with 0.3% Triton X-100 for 30 min. Slides were incubated with primary antibody, anti-SEPTIN12 (1:100, Abnova, H00124404-M), or anti-PLCζ (1:100, Abcam, ab124446), overnight at 4°C, and then detected by Alexa Fluor 488-AffiniPure Goat Anti-Mouse IgG (Jackson, 115-545-003) or Alexa Fluor 488-AffiniPure Goat Anti-Rabbit IgG (111-545-003). Hoechst 33342 was used for nucleus staining. Images were captured by Zeiss microscope with CCD.
For acrosome staining of tissue sections, after blocking in 5% BSA, slides were incubated with Alexa Fluor 488-lectin-PNA (20 μg/ml, Invitrogen, L21409) for 30 min, and then washed by PBS for three times. For acrosome staining of spermatozoa, slides were fixed by 4% PFA, permeabilized with 1% Triton X-100 for 5 min, incubated with 20 μg/ml Alexa Fluor 488-lectin-PNA at 37°C for 30 min, and then washed by PBS.

The primary antibodies used for Immunofluorescence staining in this study were anti-CD31 (1:300, Abcam, ab222783), anti-ZO-1 (1:300, Abcam, ab221547), anti-Occludin (1:300, Abcam, ab216327), anti-NeuN (1:300, Abcam, ab104224), anti-GFAP (1:1000, Abcam, ab7260), anti-Sox17 (1:100, Santa Cruz Biotechnology, sc-130295), and anti-CD68 (1:300, Abcam, ab283654). The secondary antibodies used for immunofluorescence staining in this study were Alexa Fluor® 488 AffiniPure™Goat Anti-Rabbit IgG (H + L)(1:300, Jackson, 111-545-003) and Alexa Fluor® 594 AffiniPure™Goat Anti-Mouse IgG (H + L)(1:300, Jackson, 115-585-003). Primary antibodies used for western blot studies were anti-ZO-1 (1:1000, Abcam, ab221547), anti-Occludin (1:1000, Abcam, ab216327), anti-β-actin (1:2000, Servicebio, Wuhan, China), anti-Sox17 (1:500, Santa Cruz Biotechnology, sc-130295; 1:1000, Proteintech, Wuhan, China, 24903-1-AP), anti-CD31(1:1000, Abcam, ab222783), anti-UCHL1 (1:1000, Abcam, ab108986), anti-Flag-Tag (1:2000, Cell Signaling Technology, #14793), anti-Myc-Tag (1:2000, Cell Signaling Technology, #9402), and anti-HA-Tag (1:2000, Cell Signaling Technology, #3724). The secondary antibodies used for western blot studies were HRP conjugated Goat Anti-Rabbit IgG (H + L) (1:5000, Servicebio, GB23303) and HRP conjugated Goat Anti-Mouse IgG (H + L) (1:5000, Servicebio, GB23301).

The brain sections were incubated overnight at 4°C in a humidified box with primary antibody Iba-1 (1:250). Then, the samples were incubated with secondary antibody: Alexa Fluor^® 488 AffiniPure Goat Anti-Rabbit IgG (Jackson; 1:200) for 2 h in the dark at room temperature. The following antibodies CD16/32 (1:250) and CD206 (1:250) were used overnight at 4°C. Next, the fluorescent secondary antibody Alexa Fluor^® 488 AffiniPure Donkey Anti-Goat IgG (1:200) was incubated for 2 h at room temperature in the dark at room temperature. In addition, DAPI was used to counterstain nuclei for 10 min in the dark. Finally, the sections were photographed using a laser confocal microscope (Leica TCS SP5, Germany). The merged yellow fluorescence intensity was detected by ImageJ software. And the relative quantification of each groups were based on fluorescence intensity.

The paraffin-embedded skin sections were blocked with 10% normal goat serum albumin and incubated with mouse anti-iNOS antibody (1:200 dilution, Abcam, Cambridge, MA, USA) or rabbit anti-CD206 antibody (1:200 dilution, Abcam), and rabbit anti-F4/80 antibody (1:200 dilution, Abcam) overnight at 4 °C. The sections were incubated with Alexa Fluor 488 AffiniPure goat anti-rabbit IgG (1:500 dilution, Jackson ImmunoResearch, West Grove, PA, USA) or Alexa Fluor 488-conjugated AffiniPure goat anti-mouse IgG (1:500 dilution, Jackson ImmunoResearch) and Cy3-conjugated AffiniPure goat anti-rabbit IgG (1:500 dilution, Jackson, USA) at for 1 h. Then nuclei were stained with 6-diamidino-2-phenylindole. The sections were observed by fluorescence microscopy (Nikon, Shanghai, China) and scanned by pannoramic scan. Then the double positive cells were counted by CaseViewer software at 630 × magnification and five microscopic fields were selected for each section.

Tanshinone IIA (T4952) and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich (United States).; BPL (3-n-Butylphthalide, a marketed drug for acute ischemic stroke) (H20100041) was purchased from CSPC (China); BAY-11-7082 (a NF-κB inhibitor) (S2913) was purchased from Selleck (United States); ELISA Kit was purchased from R&D Systems (United States); superoxide dismutase (SOD) and malondialdehyde (MDA) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (China); total RNA Kit was purchased from Takara (Japan). Primary antibodies: NeuN (24307), Iba-1 (17198), iκB (76041), NF-κB (p65) (8242), GAPDH (5174) were purchased from Cell Signaling Technology (United States); p-iκB (ab133462) and p-NF-κB (p-p65) (ab239882) were purchased from Abcam (United States); CD16/32 (AF1460) and CD206 (AF2535) were purchased from R&D Systems (United States). Secondary antibodies: Alexa Fluor^® 488 AffiniPure Goat Anti-Rabbit IgG, Alexa Fluor^® 488 AffiniPure Donkey Anti-Goat IgG and Cy™3 AffiniPure Donkey Anti-Rabbit IgG were purchased from Jackson (United States).

Dewaxed and rehydrated slides of colon sections were boiled in citrate-EDTA antigen retrieval solution for 10 min, and were blocked with 10% normal goat serum. And then incubated with rabbit occludin primary antibody (Proteintech, 27260-1-AP, 1:200) or mouse BRD4 primary antibody (Proteintech, 67374-1-Ig, 1:100) overnight at 4°C followed by Alexa Fluor 488-Affinipure Goat Anti-Rabbit IgG (Jackson, JAC-111-545-144, 1:1,000) or Alexa Fluor 594-Affinipure Goat Anti-Mouse IgG (Jackson, JAC-111-545-144, 1:1,000). 4’,6-dia-midino-2-phenylindole (DAPI) was used for DNA counterstain.