http://www.ihcworld.com/_protocols/histology/frozen_section.htm

Procedure1. Snap frozen fresh tissues in liquid nitrogen or isopentane pre-

cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at - 80 ºC. Alternately one can use precision cryoembedding system to perform fresh tissue embedding.

2.  Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at - 80 ºC until needed. The slides can be store at -20 ºC for short term storage (within a few weeks).

3.  Before staining, warm slides at room temperature for 30-60 minutes and fix in ice cold acetone or other alternate fixatives for 5-10 minutes. Air dry for 30-60 minutes.

4.  Wash in PBS or TBS and proceed to standard staining procedur.

Principal Factors for Good Sectioning of Frozen Specimens1. The temperature must be correct for the specimen being cut.2. The microtome must be correctly adjusted and operated.3. The cutting blade must be sharp and set at the correct angle.4. The anti-roll plate must be correctly adjusted.

Recommended Temperatures for Cutting Unfixed Frozen Tissues Tissue Type Working TemperatureBrain -12 ºCLiver -14 ºCLymph Node -14 ºCKidney -16 ºCSpleen -16 ºCMuscle -20 ºCThyroid -20 ºCSkin -25 ºCBreast -25 ºCBreast with Fat -30 ºC or belowAdipose Tissue -30 ºC or belowFixed Tissue -12 ºC to -17 ºC

http://www.biolegend.com/media_assets/support_protocol/BioLegend_IHC_Frozen.pdf

Procedure guide for immunohistochemical staining on frozen tissue sections:

Prepare frozen tissue sections (steps 1-8): 1. Place a freshly dissected tissue block (<5 mm thick) on to a pre-labeled tissue base mold. 2. Cover the entire tissue block with cryo-embedding media (e.g. OCT). 3. Slowly place the base mold containing the tissue block into liquid nitrogen till the entire tissue block is submerged into liquid nitrogen to ensure tissue is frozen completely. 4. Store the frozen tissue block at -80°C until ready for sectioning. 5. Transfer the frozen tissue block to a cryotome cryostat (e.g. -20°C) prior to sectioning and allow the temperature of the frozen tissue block to equilibrate to the temperature of the cryotome cryostat. 6. Section the frozen tissue block into a desired thickness (typically 5-10 µm) using the cryotome. 7. Place the tissue sections onto glass slides suitable for immunohistochemistry (e.g. Superfrost). 8. Dry the tissue sections overnight at room temperature. Sections can be stored in a sealed slide box at -80°C for later use.

Immunostain frozen tissue sections (steps 9-28):9. Fix the tissue sections with a suitable fixative. One of the commonly used fixation methods for frozen tissue sections is to immerse the slides in pre-cooled acetone (-20°C) for 10 min. 10. Pour off the fixative and allow acetone to evaporate from the tissue sections for > 20 min at room temperature. 11. Rinse the slides in 300 ml of 10 mM phosphate buffered saline (PBS) at a neutral pH for 2 changes, 5 min each.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742633/pdf/nihms122489.pdf

Snap-frozen brain tissue sections stored with desiccant at ambient laboratory conditions without chemical fixation are resistant to degradation for a minimum of 6 months.

Brain tissue was harvested from rats (n = 5) immediately after intravenous euthanasia (1.0 mL Euthasol I.C., Delmarva Laboratories Inc, Greenland, NH). Tissues were snap-frozen at −55°C in methylbutane cooled with dry ice. The tissue samples were submerged in the cryogenic solution for 14 seconds, removed, and embedded in optimal cutting temperature (OCT) solution (Sakura Finetek, Torrance, CA). Brains were sectioned (Microm HM550 microtome/cryostat, Microm International, Walldorf, Germany) at 20 μm (used for protein extraction) and 10 μm (used for tissue staining) at −18°C. Three consecutive sections were placed onto electrostatic-charged microscope slides (Superfrost, VWR Scientific, West Chester, PA), and the slides were either dried on a microscope slide warmer (Lablyne, Melrose Park, IL) at 50°C for 45 minutes (noted as “With Heat,” or “w/h”) or merely air dried at room temperature for 15 minutes (noted as “Without Heat,” or “wo/h”). Samples were tested after being stored at the following conditions: frozen (−80°C), ambient (20°C and 45% relative humidity), and desiccated (20°C and <10% relative humidity) for 1 day, 1 week, 1 month, and 6 months. The assay matrix is summarized in Table 1, where all tests were performed in triplicate.