Immpress polymer detection kit

Paraffin-embedded tissue sections were de-waxed in isoparaffin H (Panreac) and hydrated by quick changes through serial ethanol baths. Antigen retrieval was performed by boiling in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0, in dH₂O). Samples were subjected to several washes with dH₂O and washing buffer (NaCl 8.3 g/L, Tris 1.2 g/L; pH 7.4). Endogenous peroxidase activity was saturated by incubation with 3% H₂O₂. Blocking, primary [FAAH (Abcam #ab128917, dilution 1:100), PCNA (EMD Millipore, clone PC10, dilution 1:10.000) and CXCR4 (Abcam #124824, dilution 1:500)] and secondary antibody incubations and immunodetection were performed with the ImmPRESS™ Polymer Detection kit (Vector) following the manufacturer’s instructions. Samples were finally counterstained with hematoxylin, dehydrated in ethanol, and mounted in Eukitt® Quick-hardening mounting medium (Sigma-Aldrich).

Paraffin-embedded tissue sections were sectioned for hematoxylin and eosin staining. Alternatively, immunohistochemistry was performed on paraffin-embedded tissues after antigen retrieval if necessary, by boiling for 20 minutes in sodium citrate 10 mM, Tween 0.05% (pH 6); Tris 10 mM, EDTA 1 mM (pH 9.0); or Vector Unmasking Solution (H3300, Vector Laboratories, Burlingame, California), depending on the primary antibody. After 2.5% normal horse serum blocking for 1 hour, slides were incubated overnight at room temperature, with primary antibodies at the indicated concentrations (SI Appendix S2 Table). Primary antibodies were detected with appropriate polymers (ImmPress Polymer Detection Kit, Vector Laboratories). Polymer-coupled HRP activity was then detected with either Vectastain ABC (PKD4000, Vector Laboratories) for brightfield images or Tyramide SuperBoost Kits with Alexa Fluor Tyramide for fluorescence (Invitrogen, Waltham, Massachusetts). Nuclei were counterstained with hematoxylin or Hoechst (Invitrogen). Images were acquired with a Zeiss Axioplan 2, Zeiss AxioImager with Apotome2, or Zeiss Axioscan Z1 slide scanner (Zeiss, Oberkochen, Germany). They were minimally processed for global levels and white balance using Zeiss Zen (Zeiss). Image settings and processing were identical across genotypes.

Ocular sections were stained by the standard indirect immunoperoxidase method using primary antibodies against GLP-1R (rabbit monoclonal; 1:1000; abcam, Cambridge, UK) and CD31 (rabbit monoclonal; 1:500; abcam). Antigen retrieval of the sections was performed using a microwave for 15 min in heated Tris–EDTA buffer (pH 9.0). Labeled proteins were visualized using polymeric horseradish peroxidase–conjugated secondary antibodies (ImmPRESS™ polymer detection kit; Vector Laboratories, Burlingame, CA, USA). Peroxidase activity was detected via the diaminobenzidine reaction (Liquid DAB + ; DAKO Japan, Tokyo, Japan), and sections were counterstained with hematoxylin.
To detect the localization of GLP-1R, we used the pancreatic tissue of the SD rats, which was obtained previously, as a positive control for GLP-1R. In addition, as a negative control for GLP-1R in the pancreatic tissue, the pancreatic section was stained without incubating the primary antibody against GLP-1R.
For quantification, CD31-positive areas within 1500 µm of the optic nerve disc in the retinas were measured automatically using an image analyzer (WinRoof version 6.4, Mitani Corporation, Fukui, Japan). The areas positively stained for CD31 were independently measured and expressed as ratios relative to the areas of the entire retinal regions within 1500 µm of the optic nerve disc.

To assess the histological distribution of HB-EGF in the normal and infected ME, male Sprague-Dawley rats (200–250 g) were sacrificed 3 h, 6 h, 12 h, 24 h, 2d, 3d, 5d or 7d after inoculation with NTHi as described above. Untreated animals (0 h) served as controls. After deep anesthesia with rodent cocktail (a mixture of ketamine and xylazine hydrochloride at 100 mg/mL and acepromazine at 10 mg/mL) delivered intramuscularly, rats were transcardially perfused with saline followed by 4% paraformaldehyde solution. The ME bullae were immediately removed, post-fixed for 24 h, and decalcified in solution containing 4% paraformaldehyde and 8% EDTA for 2 weeks. Specimens were then embedded in OCT compound and serial frozen sections (6 µm) were cut on a cryostat. Sections were dried overnight. Slides were washed with PBS, and endogenous peroxidase activity was quenched with 0.3% H₂O₂ for 30 min. Then, following 30 min of blocking with 2.5% normal horse serum, sections were incubated with goat polyclonal anti-HB-EGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA USA) at a concentration of 1∶50 overnight. For detection of the primary antibody, ImmPRESS Polymer Detection Kit and DAB substrate (Vector Laboratories, Burlingame, CA USA) were used. Finally, sections were counterstained with hematoxylin and mounted. Three animals were evaluated at each time point.