Immpact dab peroxidase hrp substrate sk 4105

Pancreas tissues were fixed in buffered 4% formalin for 48 h and then embedded in paraffin. Sections were deparaffinized and hydrated. Heat-mediated antigen retrieval was performed with 10 mM citrate buffer pH 6.0 (Thermo Fisher Scientific, Waltham, MA, USA). Endogenous peroxide was inhibited by incubating with a freshly prepared 3% H₂O₂ solution in MeOH. Unspecific antigens were blockaded by incubating sections for 1 h with 2.5% horse serum (VE-S-2000, Vector Laboratories Inc., Burlingame, CA, USA). For assessing the cellular structure, pancreas sections were stained with guinea pig anti-insulin (A0564, Agilent Dako, Santa Clara, CA, USA; Table 3) antibody, followed by biotinylated secondary antibody and VECTASTAIN ABC reagent (VECTASTAIN ABC-Peroxidase kit, Vector laboratories). Color was developed after incubation with 3,3′-diaminobenzidine (DAB) substrate (ImmPACT DAB peroxidase (HRP) substrate, SK-4105, Vector Laboratories Inc., Burlingame, CA, USA), followed by hematoxylin counterstaining and mounting (Vecmount H-5000, Vector laboratories Inc., Burlingame, CA, USA). Stained sections were photographed using the LSM 700 imaging system (Zeiss, Oberkochen, Germany). Panoramic images were taken for the entire section using ZEN BLUE software (Zeiss, Oberkochen, Germany).

Carboxylated polystyrene NPs of different sizes were purchased from Life technologies (Carlsbad, CA, USA). Specifically, 20 nm (F8783), 40 nm (F8793), 100 nm (F8797) and 500 nm (F8813) NPs with dark red (λ_ex/λ_em = 660/680), red (λ_ex/λ_em = 580/605), blue (λ_ex/λ_em = 350/440) and yellow-green (λ_ex/λ_em = 505/515) fluorescence, respectively, were used. Methoxypolyethylene glycol amine 2000 (mPEGamine 2 KDa) (06676), methoxypolyethylene glycol amine 750 (mPEGamine 750 Da) (07966), n-[3-dimethylaminopropyl]-n-ethyl, n-[3-dimethylaminopropyl]-n-ethyl [EDC] (E1769), MES hemisodium buffer (M8902), N-Hydroxysuccinimide (NHS) (56405), and Peroxidase type II from horseradish (P8250-50KU) were purchased from Sigma Aldrich (St. Louis, MO, USA). ImmPACT DAB peroxidase (HRP) substrate (SK-4105) was purchased from Vector laboratories (Burlingame, CA, USA). Slide-A-Lyzer Cassettes (20 K) (66003) were purchased from ThermoFisher scientific (Waltham, MA, USA). Fluorescent mounting media (Vectashield, Vector Labs, Burlingame, Ca, USA)