Hrp conjugated goat anti rabbit

Salivary gland, brain, gut, fat body and ovary proteins from female adults were extracted and homogenized in 1 mL PBS. The extract was centrifuged at 13,000 × g for 15 min at 4°C and the supernatant was collected. To prepare the hemolymph sample, total hemolymph of about 100 nymphs was collected by tearing their integument carefully in 20 μL PBS buffer containing 1x proteinase inhibitor cocktail. After centrifugation, the cell-free supernatants were further concentrated to a final volume of 500 μL using a 10 kDa ultrafiltration tube.
Protein samples were mixed with loading buffer, denatured, and separated on 4%-20% SDS–PAGE gels (GenScript, Nanjing, China). The antibodies used in the experiments included rabbit anti-NlG14 (1:5,000) custom-developed by Zoonbio (Nanjing, China), mouse anti-tubulin (1:7,500; Beyotime), HRP-conjugated goat anti-rabbit (1:10,000; Cwbio), and HRP-conjugated goat anti-mouse (1,10, 000; Cwbio). Tubulin was used to estimate the protein loading. Coomassie staining-based total proteins were used as the loading control in analyzing the proteins in the hemolymph.

Testis tissue sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3% H₂O₂ at 37 °C for 15 min. After washing with PBS, the sections were incubated with blocking buffer (5% BSA) at 37 °C for 30 min. The primary antibodies and incubation conditions used were as follows: polyclonal rabbit anti-PCNA antibody (1:300, Bioss, bs-2007R) and polyclonal rabbit anti-LC3 antibody (1:100, Bioss, bs-8878R) overnight at 4 °C. Sections were washed in PBS and incubated with the secondary antibody (HRP-conjugated goat-anti-rabbit; 1:100 in PBS, CWBIO, Beijing, China). The sections were washed with PBS and incubated with DAB. Finally, the sections were counterstained with hematoxylin, dehydrated, cleaned, and examined microscopically. Non-immune rabbit serum was used to determine non-specific staining. At least 200 seminiferous tubules were evaluated in the cross sections of each testis. The number of immunoreactive (nucleus/cytoplasm-stained brown) cells was determined.