The paper 'Investigation of Hair Follicle Structure Using Sacpic Staining" is a decent example of applied research. Deer scalp skin samples will be obtained from the non-balding areas of healthy animals. This will then be transported immersed in iced supplemented RPMI. After making 1 cm2 piece of skin and washing them thoroughly with sterile PBS, these will be stored in O. C. T. at -20° C. Investigation of hair follicle structure using sacpic staining Sacpic staining will be used to distinguish the different components of hair follicles. Upon preparation of solutions Celestine blue, picric acid/ethanol, micro-indigo carmine, Safranin, and Scott’ s tap water, the frozen sections will be fixed using ice-cold acetone for 15 min.
After washing twice with distilled water (Dh2O), the fixed section will be stained with the following: the mordant Celestine blue (5 min. ), Gill’ s hematoxylin (5 min. ), Scott’ s tap water (2 min. ), and 2% Safranin (5 min. ), with washings using tap water in between. The sections will then be rehydrated with 95% ethanol, 70% ethanol, and water for 1 min. each. After which, the sections will be stained with picro-indigo carmine for 1 min.
Upon rinsing with tap water, the sections will now be dehydrated in ascending ethanol solutions (75% to absolute ethanol) for 5 min. each time. Finally, before mounting the sections using coverslips and his mount, they will be cleared using his clear: ethanol (50:50 v/v) and absolute histoclear for 4 min. each. Localization of specific molecules by immunochemistry Optimization of solutions Immunohistochemistry will be used to pinpoint specific antigens on the slide. Initially, the concentration of reaction, blocking, and diluting solutions will be optimized. Blocking of endogenous peroxidases After fixing the frozen sections with 4° C acetic acids for 10 min.
and washing three times with sterile PBS, sections will be circled with a Vector ImmEdge Pen (Vector Laboratories, Peterborough, UK) to produce a hydrophobic ring that will limit the volume of antibody applied. The sections will then be immersed in 200 µ l/section 3% (v/v) hydrogen peroxide in methanol, and the set-up will be stored in 20° C for 30 min. to deactivate endogenous peroxidases. Blocking of non-specific protein binding After washing the sections with sterile PBS for 5 min, the sections will be incubated in 5% normal mouse serum in PBS for 20 min.
Then, after blotting excess serum away, the sections will be added with 200 µ l/section rabbits polyclonal anti-SUR1 antibody diluted 1:50 with 1.5% normal mouse serum/PBS. KATP channel subunit immunohistochemistry After washing twice the blocked sections with sterile PBS for 10 min, these will be incubated with 100 µ l ExtrAvidin for 30 min. The sections will again be washed with PBS for 10 min. twice, and they will be incubated in AEC substrate solution for 25 min while being observed under the microscope.
After washing the sections with distilled H2O, the sections will be stained with Harris hematoxylin and Scott’ s tap water, with tap water rinsing after each staining. The sections will be mounted immediately with an aqueous mountant, will be left to dry overnight, and will be air-sealed with clear nail varnish.