4-6 weeks old male Wistar rats weighing 80-120 g at study initiation, were obtained/purchased from the Animal House of Ahvaz Jondishapour University of Medical
Sciences (AJUMS), Ahvaz, Iran. The rats were housed in an animal room maintained at 23 ±
3°C and a relative humidity of 30-70 % with the altering light/dark cycle of 12h during the experiment and for at least, one week prior to sensitization period (for acclimatization purposes). The animals were housed in stainless-steel wire cages in groups of 5 and had free access to PN-free standard laboratory rodent chow and water.
All protocols involving the examined animals, were orchestrated in conformity with the instructions for Laboratory Animal Experiments in AJUMS Animal Research & Care Center.
Reagents
The reagents used in this research, were purchased from the sources enclosed in parentheses: Alum [Al(OH)3] (Alhydrogel 2.0%, Serva Chemical Co., U.S.A), Cholera toxin (C-3012,
Sigma Chemical Co., St. Louis, Mo, U.S.A), DMSO (Merck Chemical Co., Germany),
Evan's blue dye (Merck Chemical Co., Germany), Rat histamine kit (LDN Chemical Co.,
Germany), K3-EDTA (Sigma Chemical Co., St. Louis, Mo, U.S.A), PBS (Merck Chemical
Co., Germany), Rat Total IgE kit (ICL Chemical Co, U.S.A).
Unshelled/incrusted crude PNs were supplied from Safee-Abad Tree-plantar Research
Station in the town of Dezfool, Khuzestan Province, Iran.
Antigen/Allergen Preparation
In the present study, PN proteins were used as allergens which were extracted from fresh crude PNs as follows, briefly:
PN bodies were pulverized by a mill and the resulted paste was defatted by n-Hexane (1:3 v/v, 3 times). Subsequent to separation process, residues were dried out and deodorized via gentle heat-treatment. Then, the obtained ...
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...id) skin testing with sterile CPE. Five min before the test, 100 μL of Evan's blue dye (5 mg/mL PBS) was injected into the tail vein to ease visualize the wheal reaction. Subsequently, 66 μL of the filter-sterilized CPE (3 mg/mL) was administered intradermally into the previously shaved abdominal skin. Negative controls
(Saline) were placed as well. A positive test response was defined as a wheal reaction showing up as a blue area/circle measuring greater than 5 mm in diameter when read at 20 min post id PN-challenge.
Detection of Vascular Leakage
Five min before intra-peritoneal PN challenge-dose adminstration, 7 rats from each group received 200 μL of Evan's blue dye (5 mg/mL PBS) by tail vein injection. Footpads (& paws) of mice were examined for signs of vascular leakage (visible blue color) 40-45 min after ip administering of 200 μg of the filter-sterilized CPE.