The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in NSD2 EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate. Keywords:Immunology, Issue 58, Gluten sensitivity, transglutaminase, autoimmunity, dermatitis, confocal microscopy, skin, rhesus monkey, Macaca mulatta Download video stream. == Protocol == == 1. Skin biopsy sample collection == Prior to skin biopsy procedure, anesthetize animals intramuscularly with 2.5 mg/kg of tiletamine hydrochloride and zolazepam hydrochloride telazol mixture (Fort Dodge Animal Health, Fort Dodge, IA). Monitor the animals from administration of anesthetic until recumbency and then remove from their enclosure. Remove the hair from the skin area of interest utilizing an Oster Golden A5 Single Speed Veterinary Clipper with a size 40 blade (Oster Professional Products, McMinnville, TN) Cruzain-IN-1 and aseptically prepare with alternating betadine scrub and alcohol. Secure a sterile fenestrated drape over the selected biopsy site. Using a sterile technique, place a 4.0 mm Miltex Punch Dermal Biopsy instrument (Miltex, York, PA) against the skin while rotating the instrument 180 degrees clockwise and counter clockwise with slight pressure until the biopsy punch transects through the dermal layers into the subcutaneous tissue. Remove the biopsy sample and grasp the transected portion of skin with forceps and free from the subcutaneous tissue. Close the skin defect with 3-0 nylon suture attached to a 3/8 circle cutting needle (Ethilon, Ethicon, Johnson & Johnson Medical Limited, Berkshire, UK) in a cruciate pattern. Give all animals 0.01 mg/kg buprenorphine hydrochloride (Hospira, Lake Forest, IL) intramuscularly for post operative analgesia. == 2. Sample processing == Work with skin biopsy samples from chronic dermatitis and healthy control rhesus macaques. Obtain two to three (4 Cruzain-IN-1 mm in diameter) biopsy samples from each animal. Fix first sample in zinc formalin (Z-fix, Anatech Ltd., Battle Creek, MI) for 24 hours, wash in water for 30 min, wash briefly in 70% ethanol, and place into ASP300 Leica tissue processor (Leica Microsystems Inc., Buffalo Grove, KS) where tissue is dehydrated with ascending grades of 70%, 80%, 95% and 100% ethanol 48 min each (Fisher Scientific, Pittsburgh, PA), followed by two changes of xylene (Fisher). Embed in paraffin media (Fisher) for long-term storage at room temperature. Place at -20oC for 20 min prior to sectioning. Prepare 6 m sections using a rotary microtome (HM325, Microm International, Waldorf, Germany). Place sections on charged slides (Fisher) and air dry at 60oC overnight. Stain with hematoxylin and eosin (H&E) standard method (described below). Fix second sample in 2% paraformaldehyde (USB Corp, Cleveland, OH) for 30 min at room temperature, wash three times in phosphate buffered saline (PBS, Gibco-Invitrogen, Carlsbad, CA), place in 30% sucrose (Fisher) for 4 Cruzain-IN-1 hours, and embed in OCT freezing medium (Sakura Finetek, Torrence, CA). Keep at -80oC for 20 min prior to sectioning. Prepare 15 m sections using the cryostat (HM560, Thermo Scientific, Kalamazoo, MI). == 3. H&E staining == Deparaffinize embedded Cruzain-IN-1 sections through three changes of Cruzain-IN-1 xylenes (Fisher) and rehydrate through graded ethanols: three changes of 100%, two changes of 95%, one change of 80% and distilled water, 2 min each. Stain with hematoxylin (Richard-Allan Scientific, Kalamazoo, MI) for one min and follow by a brief wash in running tap water. Counterstain with eosin (Sigma) for 6 min. Dehydrate stained tissues through 95% and absolute ethanol, two changes of 2 min each and then clear in three changes of xylenes, 3 min.