Biotinylated anti rabbit igg

At 4 weeks after implantation, all rats were euthanized. The oral mucosa was removed from the maxillary bone, and sections were cut on the coronal plane using a cryostat (−20 °C) after demineralization using a 5% ethylenediaminetetraacetate solution. For immunohistochemical staining, these sections were incubated with rabbit anti-Inβ4 (1:100 dilution, Chemicon International, Billerica, MA, USA) and biotinylated anti-rabbit IgG (1:100 dilution, Sigma-Aldrich, St. Louis, MO, USA), and the presence of Inβ4 was visualized using a diaminobenzidine (DAB) staining kit (Vector Laboratories, Burlingame, CA, USA), as described in a previous paper [11 (link)]. In the present study, the vertical distance between the top of the PIE or JE and the bottom of the Inβ4-positive area was measured in a direction parallel to the implant or natural tooth (Nt) surface.

For production of tissue sections, the testis was dissected from mice (90 days old), fixed in 4% paraformaldehyde for 24 h, dehydrated through a graded ethanol series, and embedded in paraffin. Tissue sections 7 mm thick were cut and mounted on glass slides pre-coated with poly-L-lysine solutions. Then the dehydrated sections were placed in citrate buffer (0 .1M citrate, 0.1 M sodium citrate; pH 6.0). Antigen retrieval was performed by heating in a microwave oven (750 W for 10 min twice) and cooling slowly to room temperature. Endogenous catalase deactivation was performed by immersion of slides in 0.3% (v/v) hydrogen peroxide in methanol for 1 h at 37 °C.
For IHC staining, the sections were washed and then incubated with 10% goat serum for 30 min at 37 °C. The sections were washed with PBS and incubated with rabbit anti-MTNR1A/1B antibody (1:200, bs-0027R/bs-0963R, Bioss, Beijing, China) overnight at 4 °C. After washing in PBS, the sections were incubated with biotinylated anti-Rabbit IgG (Sigma-Aldrich, St. Louis, USA) for 10 min at 37 °C, and then immersed in horseradish peroxidase labeled streptavidin for 10 min at 37 °C. Appropriate negative control slides were run in parallel without a primary antibody. The slides were imaged using a digital microscope (Motic, Wetzlar, Germany).

Maxisorb Immunoplates were coated with mAb 4G8 (epitope 17 to 24) (Covance, http://www.bioscience.co.uk/covance) in carbonate buffer overnight. Plates were blocked with 5% milk powder in PBS-Tween and samples were applied. The detection antibody was an Aβ₄₂ selective rabbit mAb BA3-9 (Covance) followed by biotinylated anti-rabbit IgG (Sigma). Total Aβ was visualised by addition of 1 mg/ml 4-nitrophenyl phosphate and optical density was read in a spectrophotometer at 405 nm. Absorbance was measured on a microplate reader at 405 nm and results were calculated by comparison to serial dilutions of synthetic Aβ_1–42.

Nunc Maxisorp immunoplates were coated with mAb 4G8 (epitope 17-24) (Covance) in carbonate buffer overnight. Plates were blocked with 5% milk powder in PBS-tween, and samples were applied. The detection antibody was an Aβ₄₂ selective rabbit mAb BA3-9 (Covance) followed by biotinylated anti-rabbit IgG (Sigma-Aldrich). Total Aβ was visualized by addition of the substrate (pNPP) 1 ng/mL, diluted in diethanolamine buffer, and optical density was read in a spectrophotometer at 405 nm.

Endoplasmic reticulum (ER) fractions were isolated from treated cells by using an ER preparation kit (Sigma-Aldrich) in accordance with the instructions of the manufacturer. Homogenized cell extracts were separated on a discontinuous density gradient (OptiPrep™). Serial 1-mL aliquots were collected from the bottom of gradients. Fractions were diluted 1:20 in carbonate buffer and distributed into Nunc Maxisorp immunoplates (Nunc, Roskilde, Denmark) overnight. Plates were blocked with 5% milk powder and probed with monoclonal antibody (mAb) reactive to the ER marker Grp 78 (Stressgen Biotechnology, Victoria, Canada), polyclonal anti-PAF receptor (Cayman Chemical Company, Ann Arbor, MI, USA), anti-cholesterol ester hydrolase (CEH) (LifeSpan BioSciences, Seattle, WA, USA), or anti-acyl-coenzyme A:cholesterol acyltransferase (ACAT) (Abcam, Cambridge, UK), followed by biotinylated anti-rabbit IgG (Sigma-Aldrich), extravidin-alkaline phosphatase (Sigma-Aldrich), and the addition of the substrate p-nitrophenyl phosphate (p-NPP), 1 ng/mL, diluted in diethanolamine buffer. Optical density was read in a spectrophotometer at 405 nm.

At 4 weeks after implantation, the rats were euthanized. The oral mucosa was removed from the maxillary bone and sections were cut on the coronal plane using a cryostat (−20 °C). These sections were immunohistochemically stained with rabbit anti-Ln-332 (Chemicon International., Temecula, CA, USA) and biotinylated anti-rabbit IgG (Sigma, St. Louis, MO, USA), and then visualized with a diaminobenzidine (DAB) staining kit (Vector Laboratories, Burlingame, CA, USA) according to the method outlined in a previous paper [16 (link)].

The oral mucosa from the rat was cut into sections on the coronal plane using a cryostat. For immuno-histochemical staining, these sections were stained with rabbit anti-Ln-332 (Chemicon International Inc., Temecula, CA, USA), biotinylated anti-rabbit IgG (Sigma, St. Louis, MO, USA), and visualized by a diaminobenzidine (DAB) staining kit (Vector Laboratories, Burlingame, CA, USA) as previously reported [13 (link)]. Some sections were stained with Ladewig’s fibrin stain to observe the connective tissue (Fig. 5A).Fig. 5

Soft tissue structures on the healed tooth extraction sockets filled with bone substitute. A Ladewig’s fibrin staining of the covered soft tissue structure. B Immuno-stained histology with Laminin-332. The length of distance between the edges of the epithelial surfaces