Complete IHC kit for Cryosection

Fluorescent detection:

• Thaw kit content (except Fixation buffer) to 4^oC.
• Snap freeze freshly dissected tissues with dry ice.
• Cutting 5-10 μm cryostat sections.
• Dip-fix sections into a jar filled with Fixation buffer at -20^oC for 30 min.
• Let slides air dry for 30min.
• Dilute primary antibody at 1:100 in Block solution and add to each slide (300-500 µl is sufficient to cover the section on each slide).
• Incubate the slides in the wet chamber at room temperature for 2hr.
• Take slides out of the wet chamber, drain off antibody, and wash three times with the Wash solution.
• Dilute secondary antibodies at 1: 200 in Block solution and add to each slide and put slides back into the wet chamber.
• Incubate the slides in the wet chamber at room temperature for 1hr.
• Take slides out of the wet chamber, drain off antibody, and wash three times with the Wash solution.
• Add 20 ul of Antifade-fluorescence mounting medium to the section on each slide. Place a cover glass onto the section and apply a small pressure to remove any trapped air bubble. Air-dry at room temperature for 1hr. Store glass slides at 4^oC for less than 1 month.

Enzyme-substrate detection:

• Thaw kit content (except Fixation buffer) to 4^oC.
• Snap freeze freshly dissected tissues with dry ice.
• Cutting 5-10 μm cryostat sections.
• Dip-fix sections into a jar filled with Fixation buffer at -20^oC for 30 min.
• Let slides air dry for 30min.
• Dilute primary antibody at 1:100 in Block solution and add to each slide (300-500 µl is sufficient to cover the section on each slide).
• Incubate the slides in the wet chamber at room temperature for 2hr.
• Take slides out of the wet chamber, drain off antibody, and wash three times with the Wash solution.
• Dilute secondary antibodies at 1: 200 in Block solution and add to each slide and put slides back into the wet chamber.
• Incubate the slides in the wet chamber at room temperature for 1hr.
• Take slides out of the wet chamber, drain off antibody, and wash three times with the Wash solution.
• Apply 100 ul of substrate solution to tissue section until desired color is achieved (1-10 mins).
• Rinse off substrate with PBS.
• Dehydrate sections with increasing concentrations of ethyl alcohol (70% to 100%).
• Dehydrate sections with Xylene. Air-dry in fume hood for 30min at room temperature.
• Add 100 ul of Antifade-chromogen mounting medium to the section on each slide. Place a cover glass onto the section and apply a small pressure to remove any trapped air bubble. Air-dry at room temperature for 1hr. Store glass slides at ambient temperature.