1. Materials and reagents 1. Primary antibody 2. Secondary antibody (Invitrogen) 3. 100% ice-cold acetone 4. Horse serum (SantaCruz; sc-2483) 5. Hoechst 33342 fluorescent dye (Invitrogen; H1399) 6. ProLong Gold Anti-fade reagent (Invitrogen; P36934) 7. Immunohistochemical pen (Sigma; Z377821) 8. Nail Polish (FisherScientific; 50-949-071) 9. PBS10. Formaldehyde 11. Methanol 12. TritonX-100 II. Instrument 1. -20 ° C refrigerator 2. Wet box three, step 1. Remove 7 μm freeze-dried sections from the refrigerator and immediately soak them in ice-cold acetone for 10 minutes. Note that the optional immobilization conditions depend on the antigen used and the primary antibody. Commonly used fixatives are formaldehyde, acetone and methanol. 2. Wash the slides with PBS and blot the water around the tissue to dry the slides. Carefully circle the tissue with a PAP pen. 3. The sections were blocked with 10% horse serum solution containing 0.05% TitonX-100 in PBS for 1 hour at room temperature. 4. The blocking buffer was blotted and the sections were incubated in a wet box for 1 hour with a first anti-room temperature body diluted with 2% horse serum solution in PBS containing 0.05% Triton X-100. The sections were completely submerged with a sufficient amount of antibody solution, approximately 150 microliters. Tip, dilute the antibody according to the recommended method. 5. Aspirate the primary antibody and wash 5 times with 150 μl of PBS/Triton X-100 buffer. 6. Sections were incubated in the wet box for 1 hour at room temperature with the corresponding second fluorescent antibody AlexaFluor 488/555 antibody (Molecular Probes). The secondary antibody was diluted with a 2% horse serum solution in PBS containing 0.05% Triton X-100 at a ratio of 1:300, and a sufficient amount of secondary antibody solution (about 150 μl) was used to completely submerge the sections. 7. Aspirate the secondary antibody and wash twice with 150 μl of PBS/Triton X-100 buffer. 8. Wash the sections 3 times with 150 μl of PBS buffer. 9. Incubate sections with Hoechst 33342 nuclear dye [2 mg/ml in PBS] for 2 minutes. 10. Aspirate the secondary antibody and wash 3 times with 150 μl of PBS buffer. 11. After the sections are dry, add about 2 drops of ProLongGold anti-fade reagent to each section and cover with a coverslip. Be careful to avoid damaging the tissue too far by sliding the coverslip and not creating air bubbles. Aspirate excess reagents. 12. Store in the dark for a few hours at room temperature or over-liquid to allow the sections to dry. Once the slide is completely dry, seal the edges of the coverslip with clean nail polish. The cover can be stored in the dark at -20 °C for several weeks.
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