Extravidin

Formalin fixed ovarian tissue was paraffin embedded, 5 μM sections were cut and mounted on SuperFrost plus microscopic slides. Following deparaffinization and rehydration, slides were treated with 3% (v/v) hydrogen peroxide for 10 mins to inhibit endogenous peroxide activities. Tissues were then blocked in 5% bovine serum albumin with 0.02% sodium azide for 10 min and incubated with primary antibodies overnight at 4°c. Following 1x PBS rinse, biotinylated secondary antibodies from respective species were applied for a 2-h incubation at room temperature (1:100 dilution, Sigma) with horseradish peroxidase (Extravidin, 1:50 dilution, Sigma) followed by a brief incubation with diaminobenzidine tetrahydrochloride (Sigma). Tissues were counterstained with Carazzi’s hematoxylin for 1 min, dehydrated and mounted with Permount (Sigma).
For immunofluorescence, after rehydration and blocking with 5% BSA, sections were simultaneously stained overnight with anti-CD31 and anti-NG-2. Sections were stained with secondary antibodies against anti-CD31 (Alexa Fluor®594 nm, red, 1:100) and α-SMA (Alexa Fluor®488 nm, green, 1:100) for 1 h at room temperature. Images obtained under both 594 nm and 488 nm channels using a Fluorescent microscope (Olympus) and Metamorph Imaging software (Burlingame CA).

For the IgG subclass endpoint ELISA, biotinylated anti-mouse IgG1 or IgG2a (BD bioscience) was used as the secondary Abs. After incubation and wash, alkaline phosphatase conjugated ExtrAvidin (Sigma-Aldrich, UK) was added at 1:5000 in PBS and incubated for 30 min at RT. The development and measurement of OD and the endpoint titer determination were done as before (10 (link)).
Human Ab isotype ELISAs were also performed using methodology described in detail elsewhere (70 (link)), except that plates were coated with recombinant Pfs25 produced from the Drosophila S2 cells at 2 μg/ml.

DAB-peroxidase IHC was performed as described by Kwakowsky et al. (2018 (link)). In brief, sections were washed in PBS with 0.2% Triton X-100 (PBST) before blocking for endogenous peroxidases (50% methanol and 1% H₂O₂) for 20 min, followed by three 10-min washes in PBST and incubated for 72 h in primary antibody in immunobuffer at 4°C (Table 3). Following three 10-min washes in PBST the sections were incubated for 24 h with the biotinylated secondary antibody (anti-mouse IgG-Biotin antibody produced in goat 1:1,000) in immunobuffer at room temperature (RT). The sections were then washed in PBST before incubation with ExtrAvidin (1:1,000, E2886; Sigma, St. Louis, MO, USA) in immunobuffer for 4 h at RT, followed by three 10-min washes in PBST before development in 0.05% DAB and 0.01% H₂O₂ in 0.1 M phosphate buffer. Sections were washed in PBST, mounted onto glass slides, dried, dehydrated through a graded series of ethanol, and cleared in xylene. The slides were coverslipped with DPX mountant (1019790500; Merck, Whitehouse Station, NJ, USA). The sections were imaged on either a Leica DMRB light microscope or a Leica MZ6 dissecting microscope (Wetzlar, Germany).