Envision system hrp kit

The leg samples were decalcified in 10% Ethylene Diamine Tetraacetic Acid for 4 weeks at RT and then processed for embedding in paraffin blocks. Tissues were sectioned into 5‐μm thick slices for TRAcP staining, hematoxylin and eosin (H&E) staining, and IHC. Tibia femurs sections underwent H&E staining. Stained sections were examined using an Eclipse TS100 inverted light microscope (Nikon Instruments Inc). The total number of TRAcP‐positive cells and the osteoclast surface per bone surface (Oc.S/BS) were calculated using ImageJ software. IHC analysis was performed with heat‐induced antigen retrieval in sodium‐citrate buffer (Dako). Primary antibodies used was anti‐phospho‐PI3K at 1:100. Biotinylated secondary antibody used was the EnVision + system HRP kit (Dako), and then nuclei were stained with hematoxylin.

Routine histology stains were applied to 10‐μm‐thick serial longitudinal sections; Luxol fast blue (LFB) counterstained with nuclear fast red and Bielschowsky silver impregnation to assess myelin and axon maintenance, respectively, with protocols previously described [27 (link)]. Additionally, immunohistochemistry (IHC) for myeloid (Microglia) and lymphoid lineage cells (T and B cells) was performed to appraise the immunological aspect of the disease. Briefly, sections were deparaffinized, hydrated, and endogenous peroxidase was blocked with 3% H₂O₂ in methanol. Antigen retrieval was achieved using citrate buffer (pH = 6), followed by a 10% fetal bovine serum blocking buffer in PBS. Sections were incubated overnight (O/N) with Ionized calcium‐binding adaptor molecule 1 (rabbit Iba1; 019‐19741, Wako, 1:1000), rabbit CD3e (PAB9003, Abnova, 1:200), and rabbit B220 (BS‐4818R, Bioss, 1:200) primary antibodies and the IHC reaction was visualized with the EnVision⁺ System‐HRP Kit (DakoCytomation) for Iba1, T and B cells, respectively. Lastly, positive cells were stained dark brown using 3,3’‐Diaminobenzidine (DAB) (D5637, Sigma) as chromogen, and sections were counterstained with hematoxylin (Sigma).

Immunohistochemistry was conducted to demonstrate TRAcP and OPN expression in synovial tissue. Paraffin was removed and the tissue samples were rehydrated through a xylene-ethanol series. TRAcP positive cells were stained with Dako EnVision+ System-HRP kit according to the manufacturer’s instructions using primary antibodies recognizing both TRAcP isoforms 5A and 5B (generic TRAcP) and a specific antibody for 5A isoform in 1:100 dilution. TRAcP antibodies were produced as described previously [15 (link), 27 (link)] OPN antibody (Acris Antibodies, San Diego, CA, USA) was used in 1:200 dilution. Counterstaining was done with Mayer’s hematoxylin. To eliminate the possibility of confounding by non-specific binding of secondary antibody to tissues, the protocol was done also without primary antibodies as control; no staining was seen in the negative control samples. The relative staining intensities were quantified visually using a light microscope.

For hepatic LHBS staining, paraffin-embedded tissues were pre-warmed at 65°C for 15 min and treated with xylene for paraffin removal. Antigen retrieval was performed by boiling the samples in a tissue pretreatment buffer solution (Invitrogen) for 30 min. The slides were then incubated with mouse anti-preS1 for 1 hr and assessed using the EnVision+ System-HRP kit (DAKO, Carpintena, CA, USA). For double immunofluorescence staining, the cells were fixed and stained with the indicated primary antibodies for 1 hr, followed by secondary antibodies conjugated with Alexa-488, Alexa-594, or Alexa-647 (Molecular Probes). The cells were mounted with mounting medium that contained DAPI. The images were acquired with a Zeiss LSM780 confocal microscope or an automated Leica DMI6000 inverted microscope equipped with an HCX PL FL 20x/NA0.4 objective and an EMCCD camera (Andor Luca R, Belfast, UK) as indicated. The following primary antibodies were used in this study: mouse anti-preS1 (a gift from Ningshao Xia, Xiamen University, China), rabbit anti-SNAP tag (P9310S, New England Biolabs, MA, USA), rabbit anti-STIM1 (PA5-23623, Thermo Scientific, IL, USA), and mouse anti-γ-tubulin (sc-17787, Santa Cruz, Dallas, Texas, USA).

PAM were infected with specific viruses at specified MOIs for 2 h in supplemented serum-free RPMI-1640 with 250 ng/ml TPCK trypsin (Sigma). After 2 h, cells were rinsed three times with PBS and incubated in fresh medium for a further 4 h. Quantification of infectious virus in PAM culture supernatants was conducted as previously described21 (link) which was an immuno-cytochemical focus forming assay based on infection of MDCK cells followed by immunodetection of viral nucleoprotein (NP) expression. Briefly, MDCK cells infected for 6 h were fixed in acetone methanol for 10 min followed by peroxidase treatment for 10 min and incubation with a 1:8000 dilution of primary mouse monoclonal antibody to influenza nucleoprotein (Abcam) for 40 min at room temperature. The cells were subsequently rinsed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labelled polymer for 40 min. After gently rinsing with TBS, the cells were incubated with DAB substrate-chromogen solution for 7 min (Envision + system-HRP kit, Dako). Cells positive for viral nucleoprotein were counted with an inverted microscope and the mean of positive cells in four 96-wells was used to calculate infectious focus-forming units of virus per microlitre of infection volume.