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CHAPTER 4 4.3.4 Immunohistochemistry At the end of designated recovery periods (1, 3, 5 and 7 days post-injury), animals were euthanized and perfused transcardially with 0.9% (w/v) saline followed by 4% (w/v) paraformaldehyde. Harvested spinal cords were cryoprotected for at least 24 hrs before cryosectioned at 20 μm in the horizontal plane on a Leica CM1850 cryostat. Slices around the injury epicentre were dehydrated in 70% ethanol and then rehydrated in distilled water and subsequently in 0.1M PBS. The antigen retrieval step involved incubation in Reveal-it solution (AR2002, ImmunoSolution) for 6-12 hrs at 37 °C followed by 0.1M PBS washes. Slides were blocked in 20% (v/v) normal donkey serum in 0.1M PBS with 0.1% (w/v) bovine serum albumin (BSA) for 1 hr at room temperature before the addition of primary antibodies. Primary antibodies [GFAP (1:1000, Dako Z0334); IL1β (1:200, R&D systems AF501-NA); IBA1 (1:200, Wako 019- 19741 and Abcam ab5076); UNOS (1:150, Invitrogen PA1-039)] were diluted in 0.1M PBS containing 2% (v/v) normal donkey serum and 0.1% (w/v) BSA and incubated overnight at 4 °C. Negative controls were also included where the primary antibodies were excluded. Slides were washed with 0.1M PBS followed by secondary antibody incubations at room temperature for 1-2 hrs. Secondary antibodies [donkey anti-goat Alexa Fluor 594 (1:500, Abcam ab150132); donkey anti-rabbit Alexa Fluor 488 (1:1000, Invitrogen A-21206)] were diluted in the same way as primary antibodies and followed by 0.1M PBS washes. Slides were then incubated in 1:1000 (v/v) diluted Hoechst solution (94403; Sigma-Aldrich) and then washed off using 0.1 M PBS. 4.3.5 Image acquisition and quantification Images were scanned and imaged using a Nikon A1 confocal microscope fitted with Nikon DS-Qi1 camera. In GFAP/Hoechst staining, the entire cross-section of the spinal cord at the injury level was scanned using two lasers (405 nm and 488 nm) under ×10 magnification with a z-plane of at least 10 μm in depth. In GFAP/IL1β/Hoechst, IBA1/IL1β/Hoechst, and IBA1/UNOS/Hoechst staining, six representative images were taken in the dorsal, lateral and ventral spinal cord funiculi at the injury level on both ipsilateral and contralateral sides under ×20 magnification using three lasers (405 nm, 488 nm and 561 nm) and the same z-plane configuration as above. All imaging and laser settings were kept constant for all animals for each staining. Images were then analysed offline using ImageJ v1.46r (Schneider et al., 2012). GFAP was analysed as fluorescence per % area while GFAP/IL1β/Hoechst, IBA1/IL1β/Hoechst, and IBA1/UNOS/Hoechst were analysed using Cell Counter plugin as described earlier (Hu et al., 2016). The areas of interest were defined and quantified prior to cell counting covering a minimum area of 0.1 mm2. Cell quantification is expressed as the number of cells per unit area (mm2). 4.3.6 Statistical analysis All data were expressed as mean ± SEM unless otherwise stated. Statistical analysis was carried out using R (R Core Team, 2016). General linear mixed model and Satterthwaite’s approximation 83PDF Image | Effects of Red Light Treatment on Spinal Cord Injury
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