Budget & Services – Oncology Tissue and Imaging Services

Requesting H&Es and Unstained Sections

Animal Tissue Processing

Fixation

o    Harvest the tissue and place them in their respective labeled cassettes (use ONLY pencil); place all the cassettes in a large beaker and submerge them in 10% buffered formalin for 48 hours. Cover with 2 layers of foil and place in the hood to avoid fumes, or if available use plastic containers with lids.
o    Pour the formalin into an “excess formalin” waste container (found in the hood) and using the same beaker, rinse the blocks in running tap water (cold is fine) for 5 minutes.
o    Drain the tap water and submerge the blocks in 1x PBS and store at 4^oC until processing. Can also be submerged in 70% ethanol (except brain tissue).

Size

Tissues placed in the tissue cassettes should be no thicker than 3-4mm. If placing more than one piece in a cassette they should be of relatively similar size. Do not place 2 organs together in the same cassette that are different sizes – ex . Do not put mouse liver with mouse prostate lobes. If the tumor or organ is thicker than 3-4mm or proportionately larger than other pieces going into the same cassette – slice it into thinner pieces to create tissues with a similar thickness. Some space should remain between the tissue and cassette walls, i.e. it should not be stuffed full . This helps to assure the tissues are processed uniformly.

o    Lymph nodes should always be placed in their own cassette unaccompanied.
o    Colon and small intestines should ALWAYS be flushed out, void of any material prior
to fixation and processing. This is to ensure proper fixation and sectioning of the tissue.
o    Contact Bonnie if you have questions or need assistance.

Lens Paper

Some smaller tissues and cells need to be wrapped in lens paper to assure they will not fall out of the cassette during processing: prostate lobes and lymph nodes (if necessary). There is a special technique to folding the Lens paper — read below for instructions or see Bonnie for directions.

Please dip lens paper (and biopsy pads) in water prior to use. Failure to do so, will cause tissue to stick strongly to the paper or pad during processing, which can be difficult to remove upon embedding— ultimately ruining the tissue. When folding lens paper, on a flat surface, please lay the wet lens paper flat down on a nonstick surface and center the biopsy in the middle of the paper; then, fold both ends of the paper from the bottom and top end over the biopsy one at a time, covering the biopsy in the middle. Next, fold both the left and right side of the lens paper toward the middle, covering the biopsy. Note: The folds of the lens paper will overlap, this is expected. A correct fold should resemble a square. Place folded lens paper with biopsy into tissue cassette and set in desired fixative.

Following the initial processing of the block, request 1 H&E for each block submitted.

More cuts of each block should only be requested if the initial H&E has been reviewed and the tissue appears well fixed and suitable for the staining or further experimentation. – This helps cut costs, assures that slides and precious tissue do not get wasted and also cuts down on storage of unstained sections in the -20. Some antigens degrade over time so it is best to cut only what you need when you need it.

Do not prebake the slides – this is to be done as the first step of IHC. This helps to preserve the antigenicity of the tissue(s).

Storage

BLOCKS: room temperature or 4^oC – avoid heat, direct sunlight, and dust.
Unstained Sections: -20^oC in a Ziploc bag or wrapped in parafilm to avoid condensation.

Specimen and Tissue Handling

There is no one size fits all method of tissue preservation to handle all experimental designs. Before harvesting tissue, you need to assess your experimental design to best fit the fixation method to the procedure being use to preserve the tissue. FOUR general procedures exist: snap freezing, optimal cutting temperature compound (OCT compound) media embedding, RNA stabilizing reagent, or fixative. Here are some general guidelines to help give the best quality of tissue preservation and fixation.

Using a sterile blade, bisect the specimen into 0.5 x 0.5 x 0.5 cm (or smaller) samples. Samples should not exceed 0.5 cm on one dimension, as larger sizes may cause artifacts and cracks in the blocks; size ranges from 0.5 to 0.3 cm are optimal. The forceps/needle can be used to hold the specimen while bisecting. Avoid crushing artifacts by gently but firmly securing the specimen with the needle/forceps while cutting.
In order to minimize changes secondary to autolysis, specimens must be placed on ice and/or processed as quickly as possible after resection. Ideally, specimens should be snap frozen or placed in appropriate reagent within 5 minutes from loss of vascularization
After cutting tissue from each anatomic site to the appropriate size, specimens are placed into separate sterile labeled vials or containers for snap freezing, OCT media embedding, RNA stabilizing reagent, or fixative.

Snap (or flash) freezing in liquid nitrogen

o    Flash freezing in liquid nitrogen provides excellent specimen integrity and a wide array of options for tissue analysis. Snap-freezing rather than slowly freezing reduces ice crystal formation in tissue. Do not let frozen tissue thaw until after sectioning. The specimen is placed into a sterile 1-2 ml cryovial, which is then tightly capped and submerged in liquid nitrogen for “flash freezing”. The vial is transferred from the temporary LN2 transport container to a liquid nitrogen storage tank for long-term storage.

*To Freeze Tissue: Liquid nitrogen/ Cryo-vial method
· Label tube (1.8 to 2 ml)
·  Cut tissue
·  Put in tube
·  Screw on lid
·  Plunge in liquid nitrogen.

Formalin fixation

o    Formalin Fixation is used to stabilize protein in fresh tissue, and prevent autolysis and putrefaction. Fresh tissue specimens which have been fixed in 10% buffered formalin and embedded in paraffin blocks can be sectioned, and stained with Hematoxylin and Eosin (H&E). It is not possible to prepare high quality H&E sections from snap frozen sections; hence Formalin fixed Paraffin embedded tissue (FFPE) provides the best morphological characteristics of tissue, and FFPE sections can be used for immunohistochemical analyses of protein expression.
o    Prior to the procedure, aliquot formalin to specimen containers. The volume of formalin should be a minimum of 15 times the volume of the tissue sample – e.g., 15 ml of formalin per 1 cubic cm of tissue. A 15 ml sterile centrifuge or conical tube can be used, or other capped, leak-proof container.
o    Specimens intended for formalin fixation should be processed after the completion of other fresh tissue procedures, such as flash freezing, embedding in OCT compound, and submersion in RNA stabilizing reagent.
o    The tissue sample should be trimmed such that it is approximately 0.4 cm in thickness. Specimens that exceed this dimension may not allow for adequate perfusion of the formalin.
o    The tissue should be fixed at room temperature for a minimum of 24 to 48 hours, at which point the tissue can be transferred to a histology lab for processing and embedding in paraffin.

OCT embedded sample

Embedding in Optimal Cutting Temperature (OCT) compound followed by flash freezing not only preserves DNA, RNA, and protein integrity, but also allows for sectioning of the frozen tissue. 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.

For this method place a drop of OCT into a small plastic tray, cryo-mold or weigh boat, the tissue sample is placed in the drop and oriented. If orientation is important, special care should be taken to ensure that the surface touching the face of the mold is placed properly, as this will be the side that will produce sections. The plastic tray is held with a forceps over the mouth of the liquid nitrogen transport vessel for freezing. As it begins to freeze pour more OCT onto the specimen until it is completely covered and continue to hold the tray in the vapor phase of nitrogen. Fill each cryomold with OCT by slowly and carefully filling the mold to the top. It is important to avoid formation of air bubbles, and to ensure that the top surface of the OCT compound is completely level. Avoid any uneven surfaces. After hardening of the OCT compound, the tray is placed in a pr-labeled bag or vial and transferred to a liquid nitrogen storage tank or -80 C freezer for storage.

If orientation is not important, use a sterile forceps or needle, transfer the specimen to an OCT-filled cryomold and gently submerge the tissue in the OCT until it is completely covered. None of the tissue should remain exposed. Letting this settle for 30 sec to allow the OCT to wet surface of the tissue.

o    The OCT can be hardened by holding the cryomold with a forceps over the opening of the liquid nitrogen transfer container. Freezer gloves and a face shield should be used during the procedure. Alternatively, the molds may be placed on grate that has been placed over the opening of the LN2 container for hardening. After the OCT has hardened (about one minute), place the mold into a pre-labeled specimen bag or vial and from this point over should never be allowed to warm up.
o    Another method is to float out a metal pan (like the ones you sterilize surgical tools in) on a liquid nitrogen bath (in a Styrofoam container or large Dewar). Place fresh or cryoprotected tissue into cryomold with OCT in it, and add sufficient OCT to ensure the tissue is completely covered. Place mold onto metal tray and close lid of Dewar or Styrofoam container. Let freeze. When frozen wrap in foil and place into a biopsy bag, place into -80 deg C for long-term storage.
o    Freezing tissue on Dry Ice or in the freezer is not recommended. These methods cause freezing artifacts, and desiccation of the tissue.
o    Freezing tissue too quickly by submersing into the Liquid Nitrogen will cause the blocks to crack which makes them very difficult or impossible to section. This happens because the outside tissue begins to freeze more quickly than internal portion.

RNA stabilizing reagent

RNA stabilizing agent, as specific and non-specific RNA degradation begins immediately after tissue harvest, prompt transfer of fresh specimens into an RNA stabilizing reagent is helpful to minimize changes in gene expression, and ensure accurate quantitative analysis of gene expression. This protocol is intended for use with RNA later, but similar products (RNAzol, Trizol) are widely used. Frozen specimens are not appropriate for use with this protocol.

Fees and Prices

Please note that these prices are for JHU Cancer Center members. Rates for non-cancer members affiliated with JHU are 10% higher. Non-JHU affiliates should send inquiry to Miklhail James ([email protected]). Prices are subject to change.

Histology	Price
Grossing	$3.25
Processing	$3.25
Special Processing (same day biopsy and cell blocks)	$20.00
Embedding	$3.25
Gross, Process, Embed, and H&E	$13.50
Re-embed	$4.84
Decalcification	$6.05
Unstained Slide	$3.00
Numbered Serial Sections	$3.25
Investigator supplied and labelled unstained	$2.50
Investigator supplied and labelled unstained level section	$2.83
Plus/Poly/Silane Slide	$0.61
Chemate Slide	$0.91
Initial/level/recut H&E	$3.75
H&E on Supplied Labelled Slide	$3.50
H&E on Supplied Unstained Section	$2.00
Additional H&E	$3.00
Level H&E with Revue	$4.75
Level H&E on supplied labeled slide	$4.25
Level Unstained	$3.33
Blades	$2.90
MAS & AFB	$21.00
Additional MAS & AFB	$2.00
Initial Frozen Section	$10.00
Additional Frozen Sections	$3.75
Hourly Rate for Frozen Sectioning	$55.00
Hourly Rate for Self-Cryostat usage*	$15.00
Cryostat Instruction	$10.00
Box of “+” slides	$18.00
Tissue Block Coring for Molecular Studies (up to 5 cores/block)	$6.26
Tissue Block Coring for Molecular Studies (6-30 cores/block)	$10.00
Slide Folder	$7.50
Slide Box 100	$10.00
Slide Box 25	$5.00
Slide Mailer	$0.50
Cardboard Slide Box	$2.00
Block pickup and return fee	$25.00
Hourly Rate	$50.00
Priority Rush	50% upcharge
Standard Rush	20% upcharge

Scanning and TMA Charges

Please note that these prices are for JHU Cancer Center members. Rates for non-cancer members affliated with JHU are 10% higher. Non-JHU affiliates should send inquiry to Miklhail James. Prices are subject to change and these changes will be reflected at the iLab site.

Services	Price
Routine Histo Slide Set-up and Scan 20 X	$8.50
Routine Histo Slide Set-up and Scan 40 X	$25.00
Routine Histo Slide Set-up and Scan 40 X (biopsy)	$13.00
Manual QA after scanning for Histo slides	$1.00
Routine Cytology Smear (pap / blood) Slide Set-up and Scan 20 X	$11.00
Routine Cytology Smear (pap / blood) Slide Set-up and Scan 40 X	$31.00
TMA as Histoslide	$16.00
TMA Segmented	$41.00
Rush Charges	$1.50 / Histoslide & $10.00 / TMA slide