Alexa fluor 488 goat

Meiocytes from A. suecica (JC2) were embedded in acrylamide to preserve their three‐dimensional structure and then used for the immunolocalization studies, as previously described (Nibau et al., 2020 (link); Phillips et al., 2010 (link)). Briefly, young buds (<1 mm) were harvested and fixed in 2% (w/v) paraformaldehyde, washed, macerated with a brass rod in 1% (v/v) Lipsol in buffer A and embedded in acrylamide. Embedded meiocytes were blocked and incubated with α‐ASY1 (rat, 1:500; Armstrong et al., 2002 (link)), α‐ZYP1 (guinea pig, 1:500; Higgins et al., 2005 (link)) and α‐MLH1 (1:250; Chelysheva et al., 2010 (link)) antibody solution for 24–36 h. After washing, embedded meiocytes were incubated overnight with secondary antibodies (Alexa Flour 568 goat anti‐rat, A11077, Alexa Fluor 488 goat anti‐rabbit, A32731, and Alexa Fluor 633 goat anti‐guinea pig, A21105; Invitrogen, now ThermoFisher Scientific), used at 1:500 dilution. Images were acquired using a Leica TCS SP8 confocal microscope (https://www.leica‐microsystems.com) with maximum projection of Z‐stacks and deconvolved using the built‐in lightning software. Image analysis was carried out in imaris 7.3 (Oxford Instruments, https://imaris.oxinst.com). Diakinesis and diplotene cells were identified for image acquisition based on the presence of ASY1 signal and MLH1 foci and the absence of ZYP1 signal.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton-X and blocked with 5% BSA at room temperature. Cells were incubated with anti-Phospho Histone γH2AX primary antibody (1:500) and then with secondary antibody (1:1000) anti-rabbit immunoglobulin G (Alexa Fluor™ 488 goat, A11008, Invitrogen, Carlsbad, CA, USA). Images were captured with the fluorescence microscope Olympus IX51 (400× magnification) with digital camera Olympus XM10, using the CellSens software. Quantification was performed in Image J, by measuring signal intensity and normalizing to the number of cells assessed.

Rhesus rotavirus (RRV strain) and human astrovirus serotype 8 (HAstV-8 Yuc8 strain) were multiplied in MA104 or Caco2 cells, previously cultured as mentioned above. For this, 10 and 200 μg/ml porcine pancreatic trypsin (Sigma-Aldrich®/Cat# T-4799) were added to RRV and Yuc8 respectively, during 1 h at 37 °C in order to activate viral particles. After this time, soybean inhibitor (Life Technologies®/Cat# R-007-100) was added for 5 min at RT. Then, the activated viruses were added (adsorption period) to each cell line respectively, during 1 h in a 5 % CO₂ atmosphere at 37 °C. After one wash with PBS, fresh culture medium without FBS was added to complete 10 (RRV) and 12 (Yuc8) hour post-infection (hpi) respectively, until the immunodetection test (See below).
Anti-TLP (Triple – Layered Particles) polyclonal antibodies donated by Dr. Carlos Arturo Guerrero from Universidad Nacional de Colombia or anti-Yuc8 polyclonal antibodies donated by Dr. Ernesto Méndez^† from Instituto de Biotecnología – Universidad Nacional Autónoma de México were used to detect cytoplasmic viral antigens by immunocytochemistry or flow cytometry. The conjugates and substrates used were peroxidase – goat anti-rabbit IgG (Invitrogen™/Cat# 65-6120), AEC (3-Amino-9-ethylcarbazole – Sigma-Aldrich®/Cat# A5754) with hydrogen peroxide 0.02 %, and Alexa Fluor® 488 Goat (Life Technologies/Cat# A11034), respectively.

Paraffin sections were deparaffinised and used for immunofluorescence as previously described (Heeren et al., 2015 (link)). Primary antibodies used were rabbit anti-KRT19 (or keratin 19) (1:250; ab52625, Abcam, Cambridge, UK) and rat anti-EMCN (or endomucin) (1:150; sc-65495, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Afterwards, sections were washed in 0.05% Tween-20/PBS, treated with 0.3% Sudan Black B (Edward Gurr Ltd, London, UK) in 70% ethanol for 5 min to eliminate background autofluorescence from red blood cells (Romijn et al., 1999 (link)) and incubated with secondary antibodies diluted in blocking solution for 1 h at RT. Secondary antibodies were Alexa Fluor 488 goat anti-rabbit (1:500; A-11034, Life Technologies, Eugene, OR, USA) and Alexa Fluor 555 goat anti-rat (1:500; A-21434, Life Technologies, Eugene, OR, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Peterborough, UK) and sections were mounted in Prolong Gold anti-fade reagent (Life technologies, Eugene, OR, USA). Slides used for isotype controls were treated as above using rabbit immunoglobulin fraction (1:250; X0903, Dako, Heverlee, Belgium) and rat IgG2a (1:150, MAB006, R&D Systems, Minneapolis, MN, USA) instead of the primary antibodies.