Paraffin Processing:

Most experimental samples will require fixation procedures prior to processing. Proper fixation typically facilitates immunohistochemical analyses if desired and thus saves time and is a rather important step for histological processing. Typical fixation for soft tissues (heart, liver, kidneys, spleen, brain, etc.) a phosphate buffered 4% paraformaldehyde fixative is used in at least a 10 volume excess for 2-4 h for tissues of less than 4mm thickness. Post-fixation rinsing in PBS helps select a defined fixation time. Larger tissues can be fixed for a short time ~ 30 min. and then trimmed into smaller pieces or bissected to access internal areas.

Specialized fixation procedures and processing may be required for certain tissues (i.e. bone de-calcification) or preserving specific target antigens.

Samples should then be transferred into histology cassettes (Shandon Lipshaw cat.# - 1000957, $57.00 /500. The cassettes should be labeled with a simple numbering system, date and initials in pencil. If only small pilot studies are being performed cassettes are available in histology core. The cassettes should remain immersed in either fixative or PBS prior to dropping off to prevent tissue drying. Vessel with cassettes should be clearly marked with name, PI, extension, room and the solvent (PBS, etc.).

Cryo-Histology:

Preparation of frozen tissue for cryo-sectioning will greatly depend on desired utility of the sections. Many tissues obtained from surgical or experimental procedures are flash frozen in liquid nitrogen. These samples can be fractured into small fragments or directly embedded into OCT embedding medium on the cryostat or on dry-ice blocks within laboratory hoods. Tissue architechture and ease of sectioning will greatly depend upon the tissue type and sample size. Tissues with even densities usually work best. Removal of excess liquid prior to freezing will prevent fracturing during sectioning. Rinsing any excess blood/liquids with saline or PBS followed by absorption with Kim-wipe, gauze or paper can make a dramatic difference in the time consuming sectioning process later on and only takes a few more seconds. Cryosectioning can be followed by air drying or post-fixation procedures such as cold methanol:acetone (1:1), 10 mins. to assure no tissue loss during rigorous in situ or IH procedures.

An alternative and highly versatile procedure for frozen tissue preparation can be performed which is well suited to immunohistochemistry, in situ hybridization and in situ PCR techniques. Tissues are fixed for minimal periods depending upon size (1-2h with 4% PFA:PBS) followed by several PBS rinses at 4C and tissue saturation in PBS:0.3M sucrose at 4C overnight. Tissues are blotted to remove excess liquid and embedded in OCT media. These preparations are much easier to section, especially for investigators and personnel who are not cryo-sectioning every day. The maintenance of tissue architecture, antigen epitopes and mRNA/DNA integrity are the main advantages of employing this approach for diverse experimental procedures. Long-term storage of these samples has not been problematic in -80C for which both mRNA and protein analyses have been performed years afterwards.

For special applications such as micro-dissection, whole-mount procedures and GFP-applications protocol advice from Nancy Ryan and Kevin Claffey is available and encouraged. Rapid advancement of similar applications and procedures can be affected in this manner. Select protocols which investigators are currently using and feel that may be useful to others, please email as attachments (MS Word format) to us at reshist@nso.uchc.edu or claffey@sun.uchc.edu