Enhanced hrp dab chromogenic substrate kit

Tissue samples from sacrificed mice were fixed in 10% formaldehyde, dehydrated, embedded, and then cut into 4-μm-thick sections for hematoxylin and eosin (HE) staining assays. For immunohistochemical analysis, the sections were prepared according to the manufacturer’s protocol. Briefly, the slides were deparaffinized, hydrated, antigen-repaired, and then blocked in 4% BSA. CA16 antigen was detected using an anti-enterovirus 71 antibody and cross-reacted with CA 16 antibody (Cat # MAB979, Millipore) prepared by diluting 1:1000 in PBS containing 1% BSA. These slides were washed with PBST and incubated with goat poly-HRP anti-rabbit IgG antibody (Cat # AS040, AB clonal) as a secondary antibody for 35 min at 37 °C. Peroxidase activity was detected with an Enhanced HRP-DAB Chromogenic Substrate Kit (TianGen Biotech, Co., Ltd., Beijing, China). Finally, the slides were examined under a light microscope.

The total proteins of S. scabiei from rabbits were obtained using a mammalian protein extraction kit (CWBIO, Beijing, China). Purified rSsc-eno and total mite proteins were separated by 12% SDS-PAGE and then transferred onto nitrocellulose membranes (0.22 μm). After blocking for 2 h with 5% (w/v) skim milk (in Tris-buffered saline) at room temperature, the membranes were incubated with S. scabiei-infected rabbit serum and anti-rSsc-eno rabbit IgG (1:200 v/v) respectively overnight at 4°C, while non-infected rabbit serum and IgG purified from pre-immunized rabbit serum were used as negative controls. After that, the membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:3,000 dilutions; Boster Bio-project Co, Wuhan, China) for 1 h at 37°C. Finally, the nitrocellulose membranes were visualized using an Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, Beijing, China).

The production and purification of rAlv protein was produced with hexahistidine (His₆) tag in the N-terminus but lacking in the signal peptide with primers Alv-p-F and Alv-p-R, and then was applied to 12% SDS-polyacrylamide gel. The target stripe was cut off from the gel, which was stained with Coomassie brilliant blue before. After the standard immunization procedure against New Zealand rabbit, the polyclonal antibodies were raised. The specificity of the antibodies was tested by Western blot against the purified protein with an enhanced HRP-DAB chromogenic substrate kit (TIANGEN Biotech Co., Ltd, China).

A Mammalian Protein Extraction Kit (Solarbio, Beijing, China) was used to extract crude proteins from adult worms. Proteins were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane using an electrophoretic transfer cell (Bio-Rad, Hercules) for 30 min. After washing with TRIS-buffered saline containing Tween-20 (TBST), membranes were incubated with 5% (w/v) skimmed milk at 37 °C for 2 h and incubated at 4 °C with polyclonal antibodies (1:200 v/v dilution) or serum from infected goats (1:200 v/v dilution) for 12 h, then with a 1:1,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG (Boster, Wuhan, China) for 2 h. Signals were measured using an Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen).

Carrying on in vivo suppression assays, the other half of spleen tissue was fixed immediately in 4% paraformaldehyde, and tissue sections were deparaffinized with xylene and rehydrated through graded ethanol washes. Heat mediated antigen retrieval was conducted by boiling in Tris/EDTA buffer (pH 9.0) for 5 min. The slides were incubated with 3% hydrogen peroxide for 20 min to eliminate endogenous peroxidase and then with 10% normal goat serum at room temperature in order to block any non-specific binding. After removing excess blocking buffer, the following primary antibodies, concerning rabbit anti-human CD8 antibody (ab93278, Abcam, Cambridge, UK) and mouse anti-human CD4 antibody (ZM-0418, ZSGB-BIO, Beijing, China), were diluted to the manufacturer’s recommendations and incubated in a humidified chamber at 4 °C overnight. The HRP-conjugated goat anti-rabbit IgG antibody (PV-6001, ZSGB-BIO, Beijing, China) and goat anti-mouse IgG antibody (PV-6002, ZSGB-BIO, Beijing, China) were used as the secondary antibody at 37 °C for 30 min. Finally, staining of the tissue sections was performed with an enhanced HRP-DAB chromogenic substrate kit (TIANGEN Biotech CO., LTD., Beijing, China). The sections were then counterstained with hematoxylin and visualized under a light microscope (Nikon, Tokyo Japan).

Recombinant Eg-LAP and the total protein extracts of PSCs were detected by 12% SDS-PAGE and then transformed onto a nitrocellulose membrane. After blocking with 5% skim milk, the membranes were incubated with infected sheep serum, or rabbit anti-rEg-LAP IgG (1:200) at 37 °C for 1 h and then incubated with rabbit anti-sheep or goat anti-rabbit IgG (H + L) HRP conjugate (1:2000, Bio-Rad) for 1 h at 37 °C. An Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, Beijing, China) was used for detection. Non-infected sheep and pre-immunized rabbit sera were used as negative controls.

After SDS-PAGE (12%), recombinant proteins were transferred to nitrocellulose (NC) membranes (0.2 μm, Bio-Rad). The NC sheets were blocked in 5% skim milk in Tris-buffered saline (TBS) for 2 h at room temperature. After three washes using TBS with Tween-20 (TBST), the membranes were incubated with the serum from naturally infected giant pandas diluted by TBS (1:100) overnight at 4°C. At the same time, negative controls were performed with serum from B. schroederi-free giant pandas. Following washing steps, horseradish peroxidase (HRP)-conjugated rabbit anti-panda IgG (Zen-bioscience Co., Chengdu, China) at a dilution of 1:2000 was applied to the membranes for 2 h at room temperature. The signals were revealed using an Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen).
Total proteins of B. schroederi worms were extracted in an NP-40 cell lysis buffer (Boster, Wuhan, China). All procedures for analyzing total proteins were the same as above, except the serum was replaced by purified rBs-FABP-IgG or rBs-GAL-IgG (or naïve rabbit serum), and the secondary antibody was replaced by HRP-conjugated goat anti-rabbit IgG (Boster).