Streptavidin hrp

A rabbit and a rat anti-hGAA polyclonal antibody were produced in our laboratory by subcutaneous immunisation with recombinant human GAA (rGAA, Myozyme®, Genzyme) in complete Freund adjuvant followed by boosters in incomplete Freund adjuvant. After serum immunoglobulin’s purification (Ig-Adem kit, Ademtech), the specificity of our antibodies was checked by western blot analysis by detection of rGAA at 110 kD. The proteins in tissue extracts were separated by SDS-PAGE gel electrophoresis, and the rat purified antibody was used to blot GAA in the organs of AAV-treated Pompe mice and PBS-treated mice as negative controls. Detection was performed with a secondary anti-rat antibody coupled to AlexaFluor®680 (Life Technologies) and the Odyssey infrared imaging system (LI-COR Biotechnology Inc.). For sandwich ELISA, plates were coated with purified rabbit anti-GAA antibodies, tissue extracts were incubated, and rat anti-GAA antibodies revealed GAA. Horseradish peroxidase (HRP) conjugated donkey anti-rat IgG (1:5000, r712–035-150; Jackson Immuno Research) followed by Streptavidin/HRP (1:1000, P0397; DakoCytomation) was added and 3,3′,5,5′- Tetramethylbenzidine (TMB, BD Biosciences) was used as substrate. Reactions were stopped with 2 N H2SO4 and reading was determined at 450 nm. Quantification was done using serial dilutions of rGAA (Myozyme) as standard.

Cell lysates (10 μg) or purified podoplanin (0.1 μg) were boiled in SDS sample buffer (Nacalai Tesque, Inc., Kyoto, Japan)26 (link). The proteins were electrophoresed on 5–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd.) and were transferred onto a PVDF membrane (EMD Millipore Corp., Billerica, MA). After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the membrane was incubated with primary antibodies or biotinylated lectin (1 μg/ml; Vector Laboratories Inc., Peterborough, UK), then with peroxidase-conjugated secondary antibodies (Dako; 1/1,000 diluted) or streptavidin-HRP (Dako; 1/1,000 diluted), and developed with the ECL-plus reagent (Thermo Fisher Scientific Inc.) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Mouse plasma (25 μl) was incubated for 1 h with either a polyclonal affinity-purified goat anti-mouse FD antibody (2·8 μg, product no. AF5430; R&D Systems, Abingdon, UK) or an immunoglobulin (IgG) fraction of polyclonal goat anti-mouse C3 (2·8 μg, product no. 55463; MP Biomedicals). Samples then were mixed with 12 μl protein A/G PLUS agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The reaction was incubated overnight at 4°C. Beads were washed with PBS and sample denatured at 100°C for 5 min with glycoprotein denaturing buffer (New England Biolabs, Hitchin, UK). Denatured reaction was incubated with peptide-N-glycosidase F (product no. P0704S; New England Biolabs) for 1 h at 37°C. The samples were centrifuged and the supernatant separated. FD was detected using Western blot with a biotinylated anti-mouse FD polyclonal antibody (product no. BAF5430; R&D Systems) and streptavidin–HRP (product no. P0397; Dako, Glostrup, Denmark). The proteins were visualized using Pierce ECL Western blotting substrate (Thermo Scientific).

Paraffin-embedded liver specimens were cut in 4 μm sections and Kupffer cells were quantified by immunostaining for CD68. For that, liver sections were incubated overnight at 4°C with the primary antibody rabbit IgG-CD68 (1 : 800, Abcam, USA) and washed with phosphate buffer Tween 20; then universal biotinylated link and streptavidin-HRP were added (Dako, USA) and revealed with DAB kit (Dako, USA). The slides were counterstained with hematoxylin. The number of CD68⁺ cells was counted in 5 randomly selected HPF (×400) per slide.

Immunohistochemistry assays were performed as described previously (20 (link)). Briefly, paraffin sections were deparaffinized by xylene, rehydrated with gradient ethanol, and subjected to antigen retrieval. After H₂O₂ treatment and blockage with 10% normal goat serum, the slides were incubated with anti-NCAPG antibody (1:200; Proteinch, Shanghai, China), followed by incubation with biotinylated secondary antibody and streptavidin-HRP (Dako, K5007).

Immunohistochemistry was performed as described previously (Xu et al., 2020 (link)). Briefly, paraffin sections were deparaffinized by xylene, rehydrated with gradient ethanol, and subjected to antigen retrieval. After H₂O₂ treatment and blockage with 10% normal goat serum, the slides were incubated with indicated antibodies (Proteintech, BIRC5 Polyclonal Antibody, Rabbit Polyclonal,Catalog number: 10508-1-AP, 1:200) followed by an incubation with biotinylated secondary antibody and streptavidin-HRP (Dako, K5007).

Airway sections were formalin fixed and paraffin embedded prior to immunohistochemical staining. Sections were deparaffinized, rehydrated, processed for antigen retrieval and incubated overnight at 4 °C with CREBBP antibody (Table 2). Sections were subsequently incubated with a biotinylated goat anti-rabbit secondary antibody (1:100, Vector Laboratories, Burlingame, CA, USA) prior to visualization with Streptavidin-HRP (Dako) and 3,3-diaminobenzidine (Dako). Slides were counterstained with Harris Hematoxylin solution (Sigma, St. Louis, MO, USA) and dehydrated before coverslipping with Cytoseal 60 medium (Richard-Allan Scientific, Kalamazoo, MI, USA).
Using the Nikon Eclipse 700 (Nikon Instruments, Melville, NY, USA) with a 60× objective and SPOT Advanced software (Diagnostic Instruments, Sterling Heights, MI, USA), five images were obtained from each section. These images were analyzed for positively and negatively stained nuclei using ImagePro Plus software (Media Cybernetics, Rockville, MD, USA).