Vector sk 4100

Sections were deparaffinized and rehydrated and then rinsed in 1 × Tris-buffered saline (TBS) for 10 min. Sections were microwaved in 10 mM citrate buffer for 5 min at 40% power in a 1,100 W oven and left to cool for 20 min. After a 10 min wash in 1 × TBS, endogenous peroxidase activity was blocked with 0.5% H₂O₂ in 1 × TBS for 10 min; 10% normal goat serum was applied for 1 hr, and then VIMENTIN (Santa Cruz, at 1:30 dilution), FACTOR VIII (Deko, at 1:500), p21 (Santa Cruz, at 1:400), TUNEL (Millipore, at 1:300), and SMA antibodies (Abcam, at 1:100 in 1% bovine serum albumin [BSA]) were applied overnight. Sections were washed, incubated for 1 hr in biotinylated antirabbit antibody, diluted in 1% BSA in TBS (Vector Labs BA-1000 at 1:200), and then for 30 min with Vectastain Elite ABC reagent. Sections were washed and incubated using a 3,3′-diaminobenzidine (DAB) substrate kit for 4 min (Vector SK 4100). Sections were counterstained with hematoxylin and dehydrated. Slides were imaged using a Nikon E800 microscope. Hematoxylin and DAB staining were separated into two images using ImageJ Fiji and cells were counted using IMARIS spot count.

Ovaries were sliced and portions fixed in 10% formalin for 24 hours and then changed into 70% ethanol until embedded in paraffin. Tissues were cut into 4 μm sections and mounted onto polylysine-coated slides. Slides were deparaffinized through three changes of xylene and through graded alcohols to water and microwaved in unmasking solution (Vector H-3300) for antigen retrieval. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide in methanol for 30 min. Sections were incubated with anti-HSL as indicated in Table 1 and subsequently an anti-rabbit ABC (Vector PK-4001) and stained using a DAB detection kit (Vector SK-4100). Slides were counterstained with Mayer’s hematoxylin, dehydrated through graded alcohols, and mounted with Fluoromount-G. Non-immune IgG from the host species was used as a control.