Cy3 labeled goat anti rat igg

Freshly collected female and male adults were washed, dehydrated, fixed, embedded and cut into cryostat sections as previous described [25 (link)]. To minimize non-specific binding, cryosections were treated with 10% normal goat serum in PBS containing 0.1% Tween 20 (PBST) for 1 h. Subsequently, cryosections were served with primary anti-rHcADRM1 IgG (1:200) or sham control IgG overnight at 4 °C. Prior to DNA staining with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Sigma-Aldrich), cryosections were then incubated with Cy3-labeled goat anti-rat IgG (1:500; Beyotime Biotechnology, Shanghai, China) at 37 °C for 1 h. Subsequently, the samples were immersed in anti-fade medium (Sigma-Aldrich) to prevent fluorescence fading for microscopic examination. Finally, the sections were imaged at 60× magnification using a LSM710 fluorescence microscope (Zeiss, Jena, Germany), and ZEN 2012 software (Zeiss) was used for the analysis of digital images.

Freshly collected adult male and female H. contortus parasites were washed in PBS. The worms were dehydrated and then embedded in TISSUE-TEK® O.C.T. compound (SAKURA Finetek, Torrance, USA). They were snap-frozen in liquid nitrogen and stored at -20 °C until further processing. Using a cryotome (CM1950, Leica Instruments GmbH, Wetzlar, Germany), 8 μm thick sections were cut and mounted on Poly-L-lysine hydrobromide glass slides. The sections were further treated as described previously [17 (link)]. Briefly, sections were washed with PBS and blocked with 5% BSA in PBST for 1 h at 37 °C to prevent non-specific binding. Thereafter, the sections were incubated with rat anti-API serum (1:300 dilution) or normal rat serum (negative control) for 1 h at 37 °C. Following by washing with PBS for 3 times, the sections were incubated with Cy3-labeled Goat Anti-Rat IgG (1:1,000 dilution, Beyotime Institute of Biotechnology, Shanghai, China). After washing with PBS, the sections were immersed with Antifade Mounting Medium (Beyotime Institute of Biotechnology, Shanghai, China) which prevents the fading of fluorescence during microscopic examination.

The mature parasites were suspended in TISSUE-TEK^® O.C.T. compound (SAKURA, Torrance, CA, USA) and snap-frozen in liquid nitrogen. By using cryotome (CM1950, Wetzlar, Germany), worms were cut into sections with 10-µm thickness and fixed on poly-l-lysine hydrobromide glass slides. Non-specific bindings were confiscated by treating the slides with 10% normal goat serum, followed by incubation with rat-anti-STP-1 antiserum (1:300 dilutions) or normal rat serum as first antibody and Cy3-labeled Goat Anti-Rat IgG (1:3,000 dilutions) as second antibody (Beyotime, Shanghai, China) at 37°C for 2 h in each step. For DNA staining, the sections were marked with 1.5 µM 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI: Sigma, MO, USA) for 5 min. Finally, Anti-Fade Fluoromount Medium (Beyotime, Shanghai, China) was used and sections were examined under confocal microscope.

Cadmium chloride, taurine, anti-LAMP2, anti-LC3B, and anti-p62/SQSTM1 were purchased from Sigma-Aldrich (202,908, St. Louis, MO, USA). DMEM and FBS were obtained from Gibco (Grand Island, NY, USA). Alexa Fluor 488-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-rat IgG, Lyso-Tracker Red (LTR), and Hanks’ Balanced Salt Solution were obtained from Beyotime (Shanghai, China); Anti-β-actin, anti-Atg7, anti-Beclin-1, anti-CTSB, and anti-rabbit IgG were obtained from Cell Signaling Technology Inc (Danvers, MA, USA). anti-LAMP2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DQ-BSA-Green was purchased from Invitrogen (Carlsbad, CA, USA). Anti-STX17, anti-SNAP29, and anti-VAMP8 were obtained from Abcam (UK). All of the other materials were of analytical grade.

Macrophage polarization (M0, M1, and M2) was identified by fluorescence staining and imaging. The three groups of cells were fixed for 30 min at room temperature in 4% paraformaldehyde (MP Biomedicals, Santa Ana, CA, United States). 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States) in PBS containing 0.1% Tween-20 was used to permeabilize the cells, after washing them with PBS thrice (5 min each). The cells were blocked with 3% BSA (Sigma-Aldrich) for 30 min at room temperature. After that, samples were allowed to incubate overnight at 4°C with primary antibodies, which included rabbit anti-iNOS IgG (Abcam, United States) and mouse anti-Arginase (Abcam, United States), Anti-CD86 Antibody (Abcam, United States), and Anti-Mannose Receptor Antibody (Abcam, United States) (Abcam, United States). Secondary antibodies included Cy3-labeled Goat Anti-Rat IgG (Beyotime, Shanghai, China), Cy3-labeled Goat Anti-Mouse IgG (Beyotime, Shanghai, China), and Alexa Fluor 488-labeled Goat Anti-Mouse IgG (Beyotime, Shanghai, China), and cells were counterstained with 6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) solution at 1 μg/ml. Images were obtained using a fluorescence microscope was used to capture the images (BX51; Olympus, Tokyo, and Japan).

The expression of native Hc-HAP protein in adult worms (females and males) was detected by immunofluorescence assay (IFA). Haemonchus contortus adults were obtained from the fourth stomach of goats and fixed in 4% tissue cell fixation solution for 12 h as previously described [12 (link)]. Samples were prepared into 4-μm paraffin sections, and antigens were repaired by microwave heating. Sections were placed in 5% BSA solution and incubated for 1 h at 37 °C, followed by incubation with rat anti-rHc-HAP serum and normal rat serum (negative control) overnight at 4 °C. The following day, after five washes with PBST wash solution, the sections were incubated with Cy3-labeled Goat Anti-Rat IgG (Beyotime, Shanghai, China) for 1 h at 37 °C, while being protected from light. After 5 washes with PBST, sections were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime), and fluorescence was observed with a laser confocal microscope (LSM 710; Carl Zeiss Spectroscopy GmbH, Jena, Germany).