Abcys

Bovine ovary sections (n = 4 from 8 cows) from a slaughterhouse were fixed, then deparaffinised, hydrated, and microwaved for 5 min in an antigen unmasking solution (Vector Laboratories Inc., AbCys), then allowed to reach room temperature. Sections (7 μm) were washed for 5 min in PBS then incubated in a peroxidase-blocking reagent for 10 min at room temperature in order to inhibit endogenous peroxidase activity (DakoCytomation). After two 5 min washes in PBS, the non-specific background was eliminated by blocking with 5% lamb serum in PBS for 20 min, followed by an overnight incubation at 4°C with PBS containing a rabbit primary antibody raised against CCL21 (1:100; Sigma, Aldrich, Saint Quentin Fallavier, France). Sections were washed in PBS twice for 5 min each, then incubated for 30 min at room temperature with a ready-to-use labeled polymer-HRP anti-rabbit antibody (EnVision Plus HRP system; DakoCytomation). The sections were washed in PBS twice, and staining was revealed after incubation at room temperature with 3,3-diaminobenzidine tetrahydrochloride (DAB) (Liquid DAB+ Substrate Chromogen System; DakoCytomation). The negative controls were prepared by replacing the primary antibodies with rabbit IgG.

Six‐micrometre transverse cryostat sections were prepared from samples frozen in isopentane and fixed and permeabilized in acetone (10 min, −20°C); and non‐specific labelling was blocked using 10% DFBS in PBS (30 min, RT). Human ALDH1A1, 1A2, 1A3, 1B1, 1L1, 2, 3A1, 3A2, 3B1, 3B2, 4A1, 5A1, 6A1, 7A1, 8A1, 9A1, and 18A1 were labelled using rabbit, goat, or mouse Ab (1/100–1/500, 2 h, RT, Table1) followed by the secondary Ab linked to a fluorophore (Alexa Fluor 568, 1/300 in PBS, 30 min at 4°C). Then sections were incubated with a rabbit polyclonal anti‐laminin Ab (1/200, 1 h, RT, Dako), followed by goat anti‐rabbit Ab (Alexa Fluor 488, 1/400, 1 h, RT) to delineate skeletal muscle fibres. In situations where the first Ab was already produced in the rabbit Ab, we first coupled the anti‐laminin Ab using the Mix‐n‐Stain CF488A antibody labelling kit according to supplier instructions (Sigma) and incubated this stained product (1/300, 30 min, RT). Nuclei were labelled with DAPI in mounting medium (Vectashield, AbCys). Negative controls were obtained by omitting primary Abs. Sections were observed using a Zeiss fluorescence microscope, images were captured using a Sony CCD cooled camera, and the Metaview® software and final figures were made using Adobe Photoshop®.