Fixation & Tissue process Protocol  (University of Pennsylvania)     

Fixative

* Fresh made RNase free 4% paraformaldehyde (4%PF) used for In Situ Hybridization.

* 4% paraformaldehyde or 10% buffered formalin used for routine histology (i.e. H&E staining)

* 2% paraformaldehyde, 4% paraformaldehyde or 10% buffered formalin used Immunocytochemistry.

Fixation Procedure

The following protocols to be used for tissue sample processed for light microscopy with H&E staining, immunocytochemistry and In Situ hybridization. RNase free PBS and fixative must be used for In Situ hybridization with RNA probe.

Use 60ml yellow cap container with 30-40 ml solutions or 20ml scintillation vial with 10-15ml solutions to fix, wash and dehydrate the samples.

Well-fixed samples can be stored in same fixative at 4ûC for several days or even longer for H&E staining. In Situ hybridization sample must be dehydrated and store at -20°C with 100% ethanol after 24-48 hours fixation. Generally Immunocytochemistry sample must be dehydrated and store at -20°C with 100% ethanol after overnight fixation.

Tissue fixation

The tissue removed from the animal should be placed into cold PBS as soon as possible.

Wash tissue with PBS to remove all blood.

Place tissue to fixative for 10-15 minutes or one hour.

Cut tissue to proper size. The size can be 2X2mm to 1X2cm, but thickness must be thinner than 3mm for better fixation. The cutting surface of the tissue should be flat and smooth.

Transfer tissue to fixative and swirl the container to ensure all tissues are completely immersed in fixative. The volume of fixative must be 20 -30times over the sample.

Fix tissue at 4ûC for 1 hours.

Change to new fixative once and leave at 4ûC for overnight.

Check sample the next day to ensure proper fixation. Trim if necessary.

Embryo fixation

After collection of embryos, wash briefly with cold PBS.

Place embryos in fixative. Fix embryos at 4 °C. Fix embryos with shaking for day 10 or larger embryos.

Change fixative once after 1-2 hours.

The duration of the fixation are different depending on the age of the embryos and method of the staining.

For In Situ:

Embryos day 7-10: Fix overnight. Fix 2 days if the embryos with uterus.

Embryos day 11-18: Fix 36-48 hours.

Embryos day 18 to New born:

* Peel off skin and cut off head and limbs if those tissues are not interested or can be embedded separately.

* Make a puncture in chest and abdominal cavity. Fix overnight.

* Place embryos to new fixative. Fix for total 48 hours.

For Immunocytochemistry:

Embryos day 7-10: Fix 2-4 hours.

Embryos day 11-18: Fix overnight

Embryos day 18 to New born:

* Peel off skin and cut off head and limbs if those tissues are not interested or can be embedded separately.

* Make a puncture in chest and abdominal cavity. Fix overnight.

Tissue Process

* The following wash and dehydration steps should be carried out at 4 °C on a shaker if the samples are processed for InSitu or immunocytochemistry.

( No shaking with Day 8.5-9.5 embryos )

* Use all RNase free solutions for InSitu.

Wash with PBS 1 min.

Wash with PBS 20 min X3.

Dehydrate:
30% ETOH in ddH2O 1-2hours.
50% ETOH in ddH2O 1-2hours.
70% ETOH in ddH2O 4 hours or O/N.
95% ETOH 3 hour X 2 or O/N.
100% ETOH 1 hour X2.
Store at -20°C.
100% ETOH 1hour X3 at RT.

Xylene 30-40 min X2, Check embryos.

Paraffin 40 min X3.

Embedding. Store at 4°C.

To obtain best morphology and staining result the following care should be taken:

Use good quality fixative and enough mount of fixative.

Use sharp razor blade and fine instruments such as scissors and forceps to

handle specimen. A piece of dental wax can be used as a cutting board.

To minimize morphology artifact, treat tissue as gently as possible.

Keep embryos in cold PBS when dissect the embryos. Put dissected embryos into fixative as soon as possible.