Goat anti rabbit igg h l alexa fluor 647

S. japonicum eggs or adult worms isolated from infected mice were fixed in 4% paraformaldehyde for 30 min, embedded in paraffin, and sectioned at 3 µm. Sections were deparaffinized with xylene for two washes of 15 min each and rehydrated in a 100, 95, 70% ethanol series and deionized water for 5 min each then put in 10 mM sodium citrate buffer (pH 6.0) (Beyotime Biotechnology, Haimen, China), which was brought to a boil and maintained at a sub-boiling temperature for 10 min for antigen retrieval. After cooling, the sections were washed three times with PBS and permeabilized with 0.03% Triton X-100 (Sigma-Aldrich) for 30 min at room temperature. Unspecific binding was blocked with 5% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. Sections of eggs and worms were incubated with the Sjp90α-1-peptide-based antibody (1:500) at 4 °C overnight. After washing with 1 × PBST, the parasite tissue sections were further incubated with goat antirabbit IgG (H+L) (Alexa Fluor^® 647, Abcam, Cambridge, UK) (1:200) for 1 h at room temperature. Cell nuclei were stained with DAPI Fluoromount (Southern Biotech, Birmingham, AL, USA) and observed under a fluorescence microscope (ZEISS, Imager.A2, Oberkochen, Germany).

The HPMECs transfected with MMP19WT and MMP19E213A cDNA were fixed with 4% paraformaldehyde (10 min), and then permeabilized with 0.1% Triton X-100 (5 min), followed by blocking with 10% goat serum (30 min), cells were incubated with anti-CD31 antibody (1:100; Abcam, Cambridge, UK, ab76533), anti-VE-Cadherin (1:100; Abcam, ab33168) and anti-α-SMA antibody (1:100; Abcam, ab240654) overnight at 4 °C, followed by incubation for 1 h at RT with Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (1:1000; Abcam, ab150113) and Goat Anti-Rabbit IgG H&L (Alexa^® Fluor 647) (1:1000; Abcam, ab150079). Cell nuclei were labeled with DAPI. Images were obtained with a Zeiss microscope.

661w cells were grown on confocal plates and fixed and permeabilized with methanol for 15 min at −20 °C, followed by three washes with PBS. Then the cells were incubated for 1 h in blocking buffer (5% normal goat or donkey serum, 0.3% Triton X-100 in PBS), following by incubation with anti-LC3B primary antibody (1:400, Cell Signaling Technology, MA, USA, Cat#83506), anti-TOM20 primary antibody (1:200, Cell Signaling Technology, Cat#42406), anti-PINK1 primary antibody (1:200, Abcam, Cambridge, UK, Cat#ab23707) and anti-Parkin primary antibody (1:200, Abcam, Cat#ab77924) overnight at 4 °C. After gently washing, the respective fluorescent secondary antibodies purchased from Abcam (Goat Anti-Mouse IgG H&L, Alexa Fluor 488, Cat#ab150113, 1:1000; Goat Anti-Rabbit IgG H&L, Alexa Fluor 647, Cat#ab150079, 1:1000; Donkey Anti-Mouse IgG H&L, Alexa Fluor 594, Cat#ab150108, 1:1000; Donkey Anti-Rabbit IgG H&L, Alexa Fluor 488, Cat#ab150073, 1:1000) were added and the cells were incubated at 37 °C for 2 h in the dark. Nuclei were stained with 0.01 mg/ml Hoechst 33342. The cells were photographed using a laser-scanning confocal microscope (Leica, Nussloch, Germany) for fluorescence and a digital camera driven by LAS AF software.

Cryosections of the enucleated eyes taken on day 3 were washed with PBS × 1, permeabilized with 1% Triton for 10 min, blocked with 5% Fetal calf serum in PBS for 60 min, and incubated at 4 °C overnight with the primary antibody, rabbit anti-IBA1 (1:500, Abcam, Cat# ab178846). The sections were washed with PBS ×1 and incubated at room temperature for 1 h with the secondary antibody, goat anti-rabbit IgG H&L Alexa Fluor 647 (1:1000, Abcam, Cat# ab150079). The sections underwent nuclear counterstaining with DAPI (Sigma Aldrich Israel, Rehovot, Israel). Images were generated using an LSM 700 inverted confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Excitation wavelengths used were 405 nm for DAPI and 647 nm for Alexa Fluor.

Cells were seeded in six-well chamber slides and treated with StemRegenin 1 (100 nM). After 48 h, the cells were washed twice with PBS and fixed for 15 min with 4% paraformaldehyde. The cells were blocked with 1% BSA for 1 h. The cells were then treated with rabbit anti-NIS (1:100; Abcam), and goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) (Abcam) diluted at 1:100, followed by DAPI.

Peritoneal macrophages were fixed with 4% paraformaldehyde for 30 minutes, then washed three times with PBS and permeabilized with 0.03% Triton X‐100 (Sigma‐Aldrich) for 30 minutes at room temperature. Unspecific binding was blocked with 5% Bovine Serum Albumin (Sigma‐Aldrich) for 1 h at room temperature. Peritoneal macrophages were incubated with rabbit anti‐mouse IRF1 (Cell Signaling Technology) (1:200) at 4°C overnight. After washing with 1 × PBST, Peritoneal macrophages were incubated with g goat anti‐rabbit IgG H&L (Alexa Fluor® 647, Abcam) (1:200) for 1 h at room temperature, respectively. Cell nucleus was stained with DAPI Fluoromount (Southern Biotech, Birmingham, USA) and observed under a fluorescence microscope (ZEISS, Imager.A2, Germany).