Cy3 conjugated affinipure goat anti rabbit igg h l

For immunofluorescence staining, tissue sections were fixed with 4% paraformaldehyde for 20 min, incubated with permeabilization solution (0.1% Triton × 100 and 0.1% sodium citrate) on ice for 2 min and blocked with horse serum for 2 h. Sections were stained with antibodies against Mac3 (1:200, Cat^# ab199947, Abcam), CD31 (1:200, Cat^# sc-376764, Santa Cruz Biotechnology), α-SMA (1:200, Cat^#sc-53142, Santa Cruz Biotechnology), or AIBP (1:200, Cat^# ab75114, Abcam) followed by detection with fluorochrome-conjugated secondary antibodies (fluorescein (FITC)–conjugated Affinipure goat anti-rabbit IgG (H + L), Cat^# SA00003-2, Proteintech; fluorescein (FITC)–conjugated Affinipure goat anti-mouse IgG (H + L), Cat^# SA00003-1, Proteintech; Cy3–conjugated Affinipure goat anti-rabbit IgG (H + L), Cat^# SA00009-2, Proteintech; and Cy3–conjugated Affinipure goat anti-mouse IgG (H + L), Cat^# SA00009-1, Proteintech). Sections were counterstained with DAPI (Cat# BS097, Biosharp), coverslipped and then scanned with a fluorescence microscope (IX70; Olympus, Tokyo). Negative controls were obtained by incubating tissue sections with the corresponding secondary antibodies alone. Images were analyzed using ImageJ 2X software (Media Cybernetics, Bethesda, MD).

LECs (5×10⁴) were seeded on coverslips in 24-well plates and cultured with endothelial cell medium at 37°C to allow adherence. Then LECs were cultured in the presence of various culture media containing different IL-8 concentrations for 24 h. After fixation in 4% paraformaldehyde for 15 min, sequential treatments with 0.5% Triton X-100 for 10 min and 4% bovine serum albumin (BSA) for 1 h at room temperature, LECs were incubated with lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1) rabbit polyclonal antibody (1:50), VEGF-C goat polyclonal antibody (1:80) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), VEGF-D rabbit monoclonal antibody (1:100; Epitomics, Burlingame, CA, USA), VEGFR-3 rabbit polyclonal antibody (1:100; Abcam, Cambridge, UK), respectively, at 4°C overnight. Afterwards, Cy3-conjugated Affinipure goat anti-rabbit IgG (H+L) (1:1,000 dilution) and Cy3-conjugated Affinipure donkey anti-goat IgG (H+L) (1:1,000 dilution) (both from Proteintech Group, Wuhan, China) were added to the corresponding samples for an additional 1 h. After counterstaining with DAPI, coverslips were observed under a laser confocal scanning microscope (LSM710; Zeiss, Oberkochen, Germany).

The kidney specimens were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Tissue microarray sections were blocked with 5% bovine serum albumin for 1 h and then incubated with the indicated primary antibodies against NRF2 (ABclonal, Wuhan, China, Cat# A0674, 1:200 dilution) and α-SMA (Proteintech, Wuhan, China, Cat# 14395-1-AP, 1:1000 dilution) overnight. After incubation, kidney sections were incubated with Cy3-conjugated Affinipure goat anti-rabbit IgG (H + L) (Proteintech, Wuhan, China, Cat# SA00009-2, 1:1000 dilution) secondary antibodies followed by staining with the 3,3′diaminobenzidine substrate. DAB (ZSGB-BIO, Beijing, China, Cat# ZLI-9017) was used for antibody reactions. The non-immune goat IgG was used as the negative control. The slides were counterstained with hematoxylin, then dehydrated and mounted. Immunohistochemistry sections were imaged using a Nikon microscope.

A total of 5 × 10⁴ cells were plated in each well of a 24-well plate (Corning). After 24 h, the cells were fixed, permeabilized, blocked and incubated with primary antibodies against FBXW7 (Proteintech). The bound primary antibody was detected using Cy3–conjugated AffiniPure goat anti-rabbit IgG (H + L) (Proteintech). Fluorescence was detected using confocal microscopy (Leica).

EPCs (5 × 10⁴ cells) were seeded on sterilized coverslips. After treatment, cells were washed three times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO4, 1.8 mM KH₂PO4) and fixed by 4% paraformaldehyde (PFA, C104188, ALADDIN, Shanghai, China) for 30 min. The cell membrane was perforated by 0.3% Triton X-100 (Biosharp, BS084, Hefei, China) for 15 min, and the cell was blocked with 5% bovine serum albumin (BSA, Solarbio, A8010, Beijing, China) for 1.5 h. The primary antibodies rabbit anti-NLRP3 (1:200, Abways Technology, Inc., CY5651, Shanghai, China), rabbit anti-PYCARD (ASC, 1:100, Abways Technology, Inc., AY0406, Shanghai, China), rabbit anti-CD31 (1:100, affinity, P16284, Changzhou, China), rabbit anti-α-tubulin (1:1000, Abways, AB0048, Shanghai, China), rabbit anti-γ-H2AX (1:100, Proteintech, 10856-1-AP, Wuhan, China) were incubated for 5 h, respectively. After triple washed by PBS, NLRP3, and CD31 were labeled by the second fluorescent antibody Cy3–conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:100, Proteintech, SA00009-2, Wuhan, China), and ASC, α-tubulin, γ-H2AX were labeled by the second fluorescent antibody Goat Anti-Rabbit IgG (H + L) Alexa Fluor 488 (1:100, Abways Technology, Inc., AB0141, Shanghai, China) for 2 h, respectively.

The expression of p21 in lungs, especially in the bronchus, was detected by immunofluorescence. After dewaxing and dehydration treatment, the lung tissue slices were added with a 5% BSA for 30 min, and then anti-p21 antibody was incubated (1:1000 dilution; Cell Signaling Technology (CST), MA, USA) overnight at 4 °C. On the following day, slices were incubated with Cy3–conjugated Affinipure goat Anti-Rabbit IgG (H + L) (Proteintech, Wuhan, China) for 50 min. At least six images were randomly taken for each section and the red staining area was calculated by CaseViewer software.
Moreover, BEAS-2B cells cultured in 24-well culture slides were fixed with 4% paraformaldehyde for 15 min and blocked with sheep serum protein mixed with 0.3% TritonX-100 for 2 h at room temperature (26 °C). The primary antibody LC3B (GTX17380, Gene Tex), TOM20 (11802–1-AP, Proteintech) and the secondary antibody were incubated according to the manufacturer’s instruction. Finally, confocal laser scanning microscopy was used to image and assess the mitophagy.