TISSUE PREPARATION FOR IN SITU HYBRIDIZATION

1. Harvest tissue and rinse in PBS or saline.
2. Immerse tissue in 4% paraformaldehyde/0.1M sodium phosphate buffer pH7.4 (recipe follows) at 4°C for 1-3hrs. Try to avoid overnight fixation if possible as this causes problems with tissue adherence on slides during the hybridization procedure.
3. Immerse tissue in sterile 15% sucrose/1xPBS (recipe follows) 3 hrs. to overnight at 4°C.
4. Embed tissue in O.C.T. (Baxter No. M7148-4), M1 (Lipshaw) or any other convenient embedding matrix for frozen sectioning in plastic embedding molds (. Tissue should be oriented in the block appropriately for sectioning (cross-section, longitudinal etc.). Note the tissue number on the block directly for reference.
5. Freeze tissue block in liquid nitrogen. Place the bottom third (aproximately) of the block into the liquid nitrogen, allow to freeze until all but the center of the O.C.T. is frozen, and allow freezing to conclude on dry ice.
6. Store tissue blocks at -70°C in a sealed container or wrapped in foil and ship on dry ice.

It is also possible to use fresh frozen tissue for in situ hybridization if the paraformaldehyde/sucrose method is not feasible. Tissues should be rinsed in saline or PBS and frozen in O.C.T. blocks in liquid nitrogen as outlined above. Although not optimal, it is also possible to use snap frozen material tissue without an embedding matrix. The fixation, sucrose, and O.C.T. steps are used primarily to improve the tissue morphology.

It is expected that the fixation times outlined above will not result in complete fixation of large pieces of tissue. However, the additional fixation step at the beginning of the in situ hybridization procedure should ensure adequate fixation of such tissues prior to hybridization.

This protocol has been used successfully on large (up to 1 cubic cm) and small (1 cubic mm) tissue samples.

4% Paraformaldehyde

Mix in a two liter flask:

200ml 0.5M NaPO4, pH 7.4
800ml depcH20
Heat to 70°C with stirring on hot plate in fume hood
Add 40g Paraformaldehyde (EM grade, Polysciences, Cat No. 0380)

Once the solution has cleared (it should take 5 minutes or less), filter with a side-arm flask, Buchner funnel and Whatman No. 2 filter paper.
Immediately pour the solution into a one liter bottle which has been packed in ice. This cools the solution quickly and prevents breakdown of the paraformaldehyde. Store at 4°C for up to two weeks.

15% Sucrose in PBS:

500ml sterile PBS
75g "RNase free" sucrose

Mix above and filter sterilize with a disposable Nalgene filtration unit type S(0.45 micron). Store at 4°C.

USE OF FISHERBRAND SUPERFROST/PLUS MICROSCOPE SLIDES FOR IN SITU HYBRIDIZATION

We use Fisherbrand SuperFrost/Plus positively-charged microscope slides (Cat. No. 12-550-15) for all of our frozen tissue sectioning and have very good tissue retention on slides after an in situ hybridization experiment. SuperFrost/Plus slides require no preparation time prior to cryosectioning and are competitive in terms of labor cost and reagent expenses.

SECTIONING OF FROZEN TISSUES FOR IN SITU HYBRIDIZATION

1. Frozen tissues prepared as described can be wrapped and stored for many years prior to sectioning, without loss of the mRNA signal. The biggest problem with stored tissue blocks is that they tend to dessicate if not properly wrapped and the O.C.T. (Optimal Cutting Temperature compound) can be difficult to cut.
2. Blocks should be removed from the -70°C freezer and allowed to equilabrate with the cryostat chamber temperature. Tissues can be cut at any convenient temperature (-15 to -35°C) as needed. Most tissues cut well at -15°C (brain, kidney, liver, vessels, muscle, etc.) however fatty or more difficult tissues (adipose tissue, skin, lung) require temperatures as low as -35°C or less to obtain good sections. Care should be taken not to touch the face of the slides but handle by the edges only. Frozen sections 5-7µm (thinner is OK but thicker, over 10µm, may present problems for visualizing mRNA in situ) should be cut, thaw-mounted onto room-temperature slides, and immediately refrozen by placing slides with sections into a slide box (VWR micro slide box #48444-003) with a single dessicant capsule (Humi-Cap see below). When the box is full, place the top on the box and store at -70°C. Sections cut and stored with dessicant are stable for in situ hybridization and immunohistochemistry for most antigens for over 5 years.