Alexa fluor 647 donkey anti mouse

For the whole-mount imaging, we anesthetized fish at the 24 hpf, 48 hpf and 5 dpf stages with Tricaine in the same manner as described above, followed by fixation with 4% PFA for 4 h at room temperature. Subsequently, the specimens were permeabilized with three 30 min washes in 100% methanol, washed with PBS supplemented with 0.1% Tween-20 (PBST) five times for 15 min, stained with the primary antibodies in blocking solution (5% normal donkey serum, 10% DMSO, 0.1% Tween-20, in PBS) for 48 h, washed five times in PBST for 30 min, stained with secondary antibodies for 24 h, washed in PBST as described above, and finally dehydrated in 100% methanol with two 30 min washes and rendered transparent with clearing solution consisting of one part benzyl alcohol and two parts of benzyl benzoate (BABB). The primary antibodies utilized were anti-acetylated tubulin (Neuronal marker, Gene Tex), anti-HuC/HuD neuronal protein and (Abcam), all diluted 1/800 in blocking solution. Alexa fluor 555 donkey anti-rabbit and Alexa fluor 647 donkey anti-mouse (all from Invitrogen) were used as secondary antibodies at a dilution of 1/1000 in blocking solution. Note that tubulin staining was punctiform at high magnification therefore only fish of the mCherry line were used for 3D reconstruction.

For immunodetection of CD20, CD3, and p24, formalin-fixed and paraffin-embedded (FFPE) sections from human lymph nodes of HIV-infected patients were dewaxed and placed in decreasing ethanol concentrations. Heat-induced epitope retrieval was performed by autoclaving at 120 °C for 15 min in a citrate buffer pH 6 (Abcam). Then, the slides were permeabilized in 1X Tris-buffered saline (TBS) (Fisher scientific) with 0.1% Triton X-100 (Sigma-Aldrich) and 1% BSA (Sigma-Aldrich) for 10 min. Subsequently, blocking was added for 2 h with 1X TBS supplemented with 10% normal donkey serum (Jackson Immunoresearch) and 1% BSA. The slides were incubated with primary antibodies overnight at 4 °C: anti-CD20 (goat-polyclonal anti-CD20 antibody, 1/100, Abcam ab194970), anti-CD3 (mouse-monoclonal anti-CD3 antibody, 1/100, Leica Biosystems NCL-L-CD3-565), or anti-p24 (mouse-monoclonal anti-p24 antibody, 1/10, Dako-Agilent M0857), diluted in TBS 1 × −1% BSA. Next, samples were washed and incubated for 1 h with the appropriate secondary antibody: Alexa Fluor 546 donkey anti-goat (Invitrogen, 712-586-150) and Alexa Fluor 647 donkey anti-mouse (Invitrogen, A-31571), counterstained with DAPI (4′,6-diamidino-2-phenylindole-dilactate, ThermoFisher), and mounted with Fluoromount G (eBioscience).

Immunofluorescence was done on 5-μm paraffin embedded sections deparaffinized by using a Tissue-Tek slide Stainer in the deparaffinization mode and then antigen retrieved using citrate buffer pH 6.0 (BioGenex, San Ramon, CA) in a microwave oven. Slides were washed 3 × in dH20 and then transferred to 1X TBS. Slides were blocked in 1X TBS, 0.3% triton, and 5% donkey serum. Primary and secondary antibodies were diluted in 1X TBS, 1%BSA, and 0.3% triton. DAPI (Invitrogen) was used to counterstain nuclei. Slides were mounted using Prolong Antifade Diamond (Invitrogen) or Aqua Polymount (Polysciences). Confocal Z-stack images were collected on a Zeiss LSM880 and 0.5 μm optical slices were obtained. Confocal acquisition settings were consistent for image quantification. Pin1 total antibody (mouse, Santa Cruz, sc-46660, 1:100), Pin1 pSer111 (rabbit, Malter Lab, 1:1000), Alexa Fluor 647 donkey anti-mouse (Invitrogen, A31571, 1:1000), Alexa Fluor 568 donkey anti-rabbit (Invitrogen, A10042, 1:1000).

Gut tissue samples were fixed in 4% paraformaldehyde and paraffin-embedded. Tissue sections (5 μm) were treated with antigen retrieval buffer at 100°C (DAKO Target Antigen Retrieval Solution pH 6.1, S2375), blocked for 1 hour at room temperature, and incubated with the following primary antibodies: anti-CD20 (monoclonal mouse anti-human; 1:100; DAKOCytomation, M0755), anti-CD3 (polyclonal rabbit anti-human; 1:100; DAKOCytomation, A0452), and anti–PD-1 (goat anti-human; 1:50; R&D Systems, Alexa Fluor 1086). After overnight incubation at 4°C, slides were washed and stained with secondary antibodies: Alexa Fluor 488 donkey anti-rabbit (Invitrogen, A21206), Alexa Fluor 555 donkey anti-goat (Invitrogen, A21432), and Alexa Fluor 647 donkey anti-mouse (Invitrogen, A31571) for 1 hour at room temperature. Cell nuclei were visualized using DAPI nuclear counterstain (Invitrogen, D1306) and by mounting slides using ProLong Gold Antifade Mountant (Thermo Fisher Scientific).

A human fetal kidney (female) at week 15 of gestation was used for immunofluorescence using the same procedure as reported previously [30 (link)]. The following primary antibodies were used: rabbit anti-UPK1A (1:35, HPA049879, Atlas Antibodies), mouse anti-KRT7 (1:200, #MA5-11986, Thermo Fisher Scientific), rabbit anti-CDH1 (1:50, SC-7870, Santa Cruz), rabbit anti-CLDN1 (1:100, #717800, Thermo Fisher Scientific), goat anti-CAV2 (1:100, AF5788-SP, R&D Systems), mouse anti-AKAP12 (1:50, sc-376740, Santa Cruz), rabbit anti-CLDN11 (1:50, HPA013166, SIGMA Aldrich), mouse anti-POSTN (1:100, sc-398631, Santa Cruz), and goat anti-SULT1E1 (1:50, AF5545-SP, R&D Systems). The secondary antibodies were all purchased from Invitrogen and diluted to 1:500: Alexa Fluor 594 donkey anti-mouse (A21203), Alexa Fluor 594 donkey anti-rabbit (A21207), Alexa Fluor 647 donkey anti-mouse (A31571), Alexa Fluor 647 donkey anti-rabbit (A31573), and Alexa Fluor 647 donkey anti-goat (A21447). The sections were imaged on a Nikon Ti-Eclipse epifluorescence microscope equipped with an Andor iXON Ultra 888 EMCCD camera (Nikon, Tokyo, Japan).