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Blood, Vol. 112, Issue 6, 2529-2538, September 15, 2008
Histopathology of experimentally induced asthma in a murine model of sickle cell disease
Blood Nandedkar et al.
112: 2529
Supplemental materials for: Nandedkar et al
Mast Cell Tryptase: Mast cell tryptase was detected in lung sections using a modified method reported previously by Fineschi et al.1 Briefly, sections were deparaffinized and rehydrated using xylene and alcohol, respectively. Endogenous peroxidase activity was inhibited by pre-incubation with H2O2 in methanol. To unmask antigenic epitopes slides were treated with Dako Target Retrieval system (Dako Cytomation, Denmark). To eliminate nonspecific adsorption of primary antibodies to tissue elements the sections were blocked for one hour with normal donkey serum. Next, the sections were incubated with the primary antibody (goat polyclonal anti-mast cell tryptase (V-13) antibody from Santa Cruz Biotechnology, Inc., CA) at room temperature for 2-3 hours and then incubated overnight at 4°C. After three washes with PBS the sections were incubated with the biotinylated secondary anti-mouse antibody (Invitrogen, Molecular Probes, Inc.) for 2 hours at room temperature followed by avidin:biotinylated-enzyme complex (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA). The slides were then washed three times with PBS and tissue antigen localized by incubation with 3, 3′-diaminobenzidine (DAB) as per the manufacturer’s instructions. After 3 min the slides were washed in distilled water, passed through an ascending alcohol row and mounted with DPX. Stain controls were made by omitting the primary antibody. The intensity of tryptase staining was quantified using MetaMorph software. Briefly, the brown DAB stain in each slide was color coded and intensities quantified under standardized conditions in 4 sections per mouse and then averaged. The values for each group were expressed as means ± standard error of the mean. REFERENCE 1. Fineschi V, Cecchi R, Centini F, Reattelli LP, Turillazzi E. Immunohistochemical quantification of pulmonary mast-cells and post-mortem blood dosages of tryptase and eosinophil cationic protein in 48 heroin-related deaths. Forensic Sci Int. 2001;120:189-194.
Files in this Data Supplement:
- Figure S1. Correlation between % eosinophil counts in the blood and eosinophils counts in sections of lungs from WT, HbA and SCD mice (JPG, 30.3 KB)
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Percent eosinophil counts from blood smears from WT, HbA and SCD without and with OVA sensitization were plotted against the number of eosinophils observed in sections of lungs from the same animal. Eosinophil counts were performed on sections stained with H&#amp;E.
- Figure S2. Mast cell tryptase expression in lungs from from unsensitized and sensitized WT, HbA and SCD mice (JPG, 154 KB)
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(A) This figure shows images of DABA staining of lungs from UnSen, LoSen and HiSen WT, HbA and SCD mice incubated with anti-mast cell tryptase. First row: Images of lung sections from UnSen (Un), LoSen (Lo) and HiSen (Hi) WT mice. Second row: Images of lung sections from UnSen (Un), LoSen (Lo) and HiSen (Hi) HbA mice. Third row: Images of lung sections from UnSen (Un), LoSen (Lo) and HiSen (Hi) SCD mice. (B) Image anlaysis of mast cell tryptase staining in sections of lungs from UnSen, LoSen and HiSen WT, HbA and SCD mice. The data demonstrate trends in mast cell tryptase expression in lungs of WT, HbA and SCD mice after LoSen and HiSen OVA treatment protocols. The variability in tryptase measurements is too great to allow for adequate comparison.
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