Aluminum potassium sulfate

The brains were perfused transcardially with fixative solution containing 4% paraformaldehyde (Sigma-Aldrich, USA) in 0.1 M phosphate buffer pH 7.4 overnight at 4°C. Then, they were infiltrated with 30% sucrose (Merck, Germany) solution for 48–72 h. Serial sections of tissues were cut frozen on cryostat (Thermo Scientific™ HM 525 Cryostat) at 10 μm thick. All sections were picked up on slides coated with 0.3% aqueous solution of gelatin containing 0.05% aluminum potassium sulfate (Sigma-Aldrich, USA). The triplicate coronal sections of the brains were stained with 0.25% cresyl violet (Sigma-Aldrich, USA), dehydrated through graded alcohols (70, 95, 100% 2x) (RCI LabScan, Thailand), placed in xylene (Merck, Germany), and mounted using DPX mountant (Merck, Germany). The evaluation of neuron density in the hippocampus was performed under Olympus light microscope model BH-2 (Japan) at 40x magnification. Counts were performed in three adjacent fields, and the mean number was calculated and expressed as the density of neurons per 255 μm².

Mice were divided into six groups (n = 3 for each group). To induce allergic asthma, the mice were sensitized by injecting 20 μg of chicken egg albumin (OVA, Sigma-Aldrich) mixed with 2 mg of aluminum potassium sulfate (Sigma-Aldrich) in 100 μL of PBS intraperitoneally (i.p.) on day 0 and 14. From day 15 to 21, different doses of PLAG (10, 50, 250 mg/Kg) or DEX (3 mg/Kg) were administrated to the mice by oral gavage daily after being emulsified by flushing thoroughly through three-way valves. On day 20, the mice were anaesthetized with i.p. injection of 2,2,2-Tribromoethanol (Avertin, Sigma-Aldrich) dissolved in tert-Amyl alcohol (500 mg/Kg) and then challenged with 100 μg of OVA in 50μl PBS intranasally (i.n.). Forty-eight-hour after OVA challenge, the mice were sacrificed by an i.p. injection of pentobarbital (Hanlim Pharm. C., South Korea), and tracheostomy was performed. To obtain bronchoalveolar lavage fluid (BALF), 0.75mL of ice-cold PBS was infused to the lung and withdrawn via tracheal cannulation two times. Immune cell populations in BALFs were analyzed by complete blood count (CBC) using a hematology analyzer, Mindray BC-5300 (China).

Mammary glands were dissected, spread on glass slides, fixed overnight in Carnoy's fixative (six parts 100% EtOH, three parts CHCl₃, one part glacial acetic acid) and stained with carmine Alum (1 g carmine (Sigma, St Louis, MO, USA) and 2.5 g aluminum potassium sulfate (Sigma) in 500 ml distilled water). Photos were taken using BX51 light microscope (Olympus, Tokyo, Japan) equipped with a digital camera (DP71, Olympus).

Female mice were killed around 10 wk, freshly dissected mammary gland tissue were flatly placed on glass slides and fixed in Carnoy's solution (70% ethanol, 20% chloroform, and 10% glacial acetic acid) for 1 hr at room temperature. The fixed glands were washed in 70% ethanol for 15 min and then rinsed in water for 5 min. The mammary glands were stained overnight at 4°C in carmine aluminum solution (1 g carmine, sigma C1022 and 2.5 g aluminum potassium sulfate (Sigma A7167) in 500 ml water). The glands were then dehydrated progressively in 70%–95%–100% ethanol, cleared in xylene for 30 min, and mounted on glass slides with Permount (Fisher Scientific, Suwanee, GA). For β-gal staining, same tissues were 0.2% paraformaldehyde in PBS (0.1 M, PH = 6.9) with 2 mM MgCl₂ and 5 mM EGTA overnight at 4°C. After washing three times with PBS, the tissue samples were incubated for 2 h at 30°C in X-gal reaction buffer containing 5 mM K-Ferricyanide, 5 mM K-Ferrocyanide, 0.02% NaDecoxycholate, 0.01% NP-40, 2 mM MgCl2 and 1 mg/ml X-gal for 24 to 48 h at 30°C. The tissues were then washed with PBS. The stained tissues were viewed under Nikon SM21500 dissection microscope and images were recorded digitally.

Mammary glands were excised carefully, mounted onto standard microscope glass slides and fixed in Carnoy's fixative (6 parts 100% ethanol, 3 parts CHCl₃, 1 part glacial acid) overnight at room temperature. Slides were washed in 70% ethanol for 15 minutes. Ethanol was changed to distilled water gradually, with a final 5 minute rinse in water. Slides were incubated in carmine alum solution (1g carmine (Sigma C1022) and 2.5g aluminum potassium sulfate (Sigma 7167) dissolved in 500ml distilled water, boiled for 20 minutes) overnight. Slides were cleared in 70%, 95%, 100% ethanol and Xylene consecutively and incubated in Xylene overnight. Next day, slides were mounted in Permount^©.