Vectachield

Non-fluorescent monochlorobimane (MCB) bounds to reduced glutathione (GSH) in a reaction catalyzed by glutathione-s-transferase and forms a fluorescent adduct whose fluorescence intensity gives a measure of GSH content. Non-treated and treated cloned embryos (18–21 embryos per group) at 16 h post-activation were incubated with 50 μM MCB in KSOM for 40 min at 37°C. Then, embryos were washed in PBS, fixed in 4% (v/v) paraformaldehyde in PBS for 15 min and mounted onto a glass microscope slide in a microdrop of Vectachield (Vector Laboratories, Inc., Peterborough, UK). Samples were examined with an Olympus Bx41 (Olympus, Hospitalet del Llobregat, Spain) epifluorescence microscope fitted with an image capture and analyzing system (Isis software version 5.4.5, Metasystems, Boston, USA). Images were acquired using the same exposure times and settings for all embryos and analyzed with ImageJ software (Image J 1.45q, Wayne Rasband, National Institutes of Health, Bethesda, USA) for fluorescence quantification. All individual nuclei were outlined for mean fluorescence intensity calculation.

Popliteal lymph nodes were placed in OCT compound, frozen on dry ice, and stored in -20°C until sectioning. 8 μm thick sections were blocked for 1 hour in 2.5% normal goat serum (Jackson ImmunoResearch) followed by incubation with directly conjugated (rat anti-CD3 AF647 clone 17A2 and rat anti-B220 AF488 clone RA3-6B2; Biolegend) and unconjugated polyclonal rabbit anti-collagen IV (Abcam) antibodies in goat serum over night at 4°C. Sections were then incubated with secondary antibody (chicken anti-rabbit AF594; Invitrogen) for 1 hour in goat serum and mounted in mounting media with DAPI (Vectachield, Vector Laboratories). Images were acquired with a confocal microscope (Zeiss LMS 800) and analysed using Fiji (ImageJ) software.