It is now well established that there are many advantages in embedding in resin rather than paraffin wax. Resin causes less shrinkage and separation of tissue layers and thinner sections can be cut. Thinner sections are sharper under the microscope.
Semi-thin sections are particularly useful in the diagnosis of renal disease. In the renal glomerulus, pathological changes previously requiring electron microscopy for diagnosis can now be revealed under the light microscope. The histology of densely cellular tissues benefits considerably from thin sections and this is particularly true of lymph nodes in the diagnosis and classification of lymphomas.
Hard dense tissues such as bone and some botanical specimens are given improved support during sectioning, preserving the juxtaposition of hard and soft tissues. For this reason many laboratories now routinely embed bone marrow trephines in resin.
Histocryl is a hydrophilic acrylic resin, simple to use and formulated specifically for light microscopists. For those laboratories currently using an acrylic resin such as HEMA glycol methacrylate or commercially branded methacrylates, no alteration need be made to their current processing schedule.
Making up Histocryl
We supply Histocryl uncatalysed. That material has an almost unlimited shelf-life at room-temperature and does not suffer from lengthy transportation. 100mL Histocryl plus 1.5g benzoyl peroxide = catalysed resin, however, it is recommended that the whole bottle is catalysed. Mix this well, a magnetic stirrer on low speed for about one hour is effective. Undissolved catalyst that is allowed to concentrate in the bottom of a container may generate heat and cause rapid polymerisation of the entire container. Catalysed resin keeps refrigerated for about a year.
Use this catalysed medium for infiltrating the tissue and it could be used for embedding and then oven-curing.
For rapid polymerisation without oven-curing, when ready to embed, add to 10mL of catalysed resin 1 drop accelerator to initiate polymerisation. The tissue will polymerise within 10-20 minutes.
- Smear accelerator onto the base of the mould.
- Add 1 drop accelerator per 10mL catalysed resin. Mix well, fill mould, add tissue.
NB The moulds should be placed in a bath of ice cold water to dissipate the heat generated by the exothermic reaction.
Brittle blocks may result from excessive heat produced during polymerisation. This could be due to too much accelerator and especially larger blocks, which may not have been cooled adequately during polymerisation. It is very important that blocks are cooled in ice cold water to dissipate heat produced during the exothermic reaction.
The rate of polymerisation can be adjusted by varying the ratio of resin and accelerator e.g.:
One drop to 10 mL freshly catalysed resin (with 1.5% benzoyl peroxide paste) 10 minutes
One drop to 20 mL freshly catalysed resin (with 1.5% benzoyl peroxide paste) 15 minutes
One drop to 25 mL freshly catalysed resin (with 1.5% benzoyl peroxide paste) 20 minutes
Our Peel-a-Way embedding moulds are well suited for resin embedding for histology.
Fixation: Most routine fixatives can be utilised with Histocryl (neutral buffered formalin is recommended), fixation time as always, depends on the type and size of tissue.
Dehydration: A graded ethanol series is the method of choice, times again are dependent on the size of the tissue. Graded acetones should not be used.
A typical dehydration schedule for a block (12 x 10 x 3 mm) on a mixer would be:
1. 70% alcohol - 30 minutes
2. 90% alcohol - 30 minutes
Two changes absolute alcohol 30 minutes each.
Infiltration: Infiltrating solution:
100mL Histocryl plus 1.5g benzoyl peroxide paste.
Mix thoroughly until solution becomes clear.
Infiltrate tissue in 2-3 changes of catalysed resin 60 minutes each or overnight, depending on tissue and size. When fully infiltrated the tissue becomes translucent.
Cutting and Mounting: Histocryl can be sectioned using a steel knife and a standard microtome, but the method of choice is the use of a motorised microtome and glass (Ralph type) knives. Sections can be obtained from 1-5um, floated onto a warm water bath picked up onto clean slides and dried on a hot plate at 60 degrees C for at least 30 minutes. Most microscopist find a thickness of 1.5 or 2um is the best compromise between sharpness and contrast.
Staining: It is not necessary to etch or remove the resin before staining. Most routine stains give good results on tissue embedded in Histocryl using standard times and temperatures, although it may occasionally be necessary to extend some staining times.
Mounting: For best results air dry sections prior to mounting. DPX or Canada Balsam are recommended mounting media.