Molecular Pathology Core Frequently Asked Questions
A. Submissions
1. Instructions and forms
2. Fill out and submit form
3. Required fieldsB. Samples
1. Trimming
2. Fixatives
3. Submitting
4. Processing/Embedding
5. Sectioning (multiple, serial, step; DNAse/RNAse free)
1. Where do I find instructions and the form?
All instructions and forms can be found on our website as well as many excellent links to all aspects of histology.
Please bookmark/make favorite this web page in your browser or create a shortcut to the forms so that the latest versions are used.
New submitters also need to complete an Account Registration. If you will require shipping, include the complete FedEx shipping address and your FedEx account number.
Please review the Fees page for a list of services or use a calculator to generate an estimate.
2. How do I fill out the form and submit it?
You must first download and save the file to your computer. If you don't do this, it won't let you fill it out or may not save the information. Either way, you won't be able to email it as an attachment. There are several ways to save this form. In Internet Explorer, click on the link and choose the option "Save" then pick a location. Or, you can right click over the link and choose "Save Target As" then pick the location.
Yellow areas are for Core staff to fill in.
Help can be found at some fields- see your WORD window's lower status bar or press F1 key.
The submission form is divided into 4 main areas:
a. Submitter information
b. Sample information
c. Services requested
d. Sample ID table
a. Submitter information
i. *Your name (First, Last names)-
Include a phone number and/or email so you can be contacted if there are any questions.
ii. *Your PI (First, Last names)- this is the person responsible for payments.
b. Sample information
i. *Total number and check box for type of sample
submitted. Wet tissue can either be already cassetted (Cassette) or to be cassetted (Wet Tissue, incurs Trim fees).
ii.. *Species- select from drop-down choices- one species per
form.
iii. *Fixative- chose from list
iv. * Samples in- tell us what your samples are currently in and also accurately label your
container by PI, your name, and solution. Remove old
fixative labels or staff need to assume that the cassettes or tissues are in fixative. If you want your sample container returned, you must let the Submission staff know. We also have many clean containers you are welcome to use.
Fixation time can be critical for most immunolocalization assays so it is very important to keep duration constant between experiments. Handle and discard fixatives according to EHS guidelines.
If samples are submitted in fixative without duration information, our normal
procedure is to keep them in fixative until the following day. The fixation start and end dates/times are optional
fields as you can keep this information in your own files. End the
fixation before submitting by transfer to either PBS or 70% ethanol. If you have just started fixation, let the submissions staff know as we can now process with formalin and return samples by the following morning.
For samples going to fixed frozen blocks, wash
1-2 times in PBS, then transfer to cold 18%
sucrose/PBS. Some labs use 20 or 30% sucrose instead of 18%. Leave in this solution overnight in the refrigerator or until
samples sink indicating full infiltration. Submit
fixed tissues or tissues in cassettes in 18% sucrose.
c.
Services requested
ii. Check box for the final block type
iii. Unstained slides- Check this box if you want unstained slides returned to you. Indicate # slides/block. The other 2 boxes are for Core staff to fill in. Do not select multiple sections or serial/step sections unless you truly want them because of the extra technical effort required, hence additional charges.iv. Special stains- 2 drop down lists are provided and a place to list a stain. If we can not run the stain, your slides can be submitted to Shands histology for staining. Please ask staff about turn around time when submitting samples for Special stains as some require a minimum number of slides (5) to be run in a batch.
v. IHC- Use check boxes. For all other IHC, you must verify availability with Dr. Ann Fu before submitting. Availability is determined by assay complexity and IHC workload. When we are unable to provide the assay, we can share our protocol with you, provide training, or refer you to the COM EM Core.
vi. Slide scanning
vii. Slide container- if you bring your own container, check that box, otherwise we will select a slide container depending on slide number. Please recycle slide trays and 100-slide plastic boxes to us and you'll receive full credit on future submissions.
vii. Special requests and other services - list anything you think would help process, embed, section, or stain your samples. O ther services- Just ask - there are too many to list.
d. Cassette/Sample ID table- Filling in this table is optional and the
listing is provided if you want us to verify cassettes IDs. We
can check for discrepancies between total number of items submitted, cassette IDs,
and other types of omissions or errors.
Keep cassette ID
very brief and use your lab files to keep more detailed information. The
cassette ID is carried forward onto slide labels and will be shortened if it is
too complex or your characters are not supported by our
printers.
List tissues by
their first 2 letters (for example, liver is listed as "Li"). If you
list the tissues and total number of pieces in the cassettes, this information
is verified during embedding. You can also attach an Excel file instead of filling out the table and such a file is a perfect
place for listing your more complex slide IDs in a separate column.
Incorrect or incomplete submission forms may delay completion of your project.
B. Preparing Samples for Submission
1. How do I trim my tissues?
Trim tissues to fit in the cassettes without being compressed by the cassette or other tissues.
Specimens should be cut thin enough that the fixative penetrates the tissue within a reasonably short time. Allow ~1mm/hr for the fixative to penetrate your tissues and a volume of ~10 times the volume of fixative to tissue. The thickness and size of a nickel is a good guide to use when trimming samples. If several tissues are submitted that vary significantly in size or hardness, sort your samples by tissue hardness and then submit in separate cassettes. Ask us about tissue cassette guidelines as we can provide an embedding guide.
The best resource for rodent organ trimming can be found at the RENI web page:
Brief harvesting and trimming instructions:
Paired organs- weighed together then one or both trimmed for histopathology. If you want to submit both, place in separate cassettes so there is no question about left versus right. Bone: Femur and sternum are harvested for bone marrow. Open each end of a femur to allow better fixation.Decalcification: After fixation, bone must be treated to remove the calcium content. We use RDO Rapid Decalcifier (Cat.#RDO01, Apex Engineering). Other decalcification solutions can be better for IHC purposes. Decal in your lab or or check the box for Core to do it.
Liver: Remove entire organ from body cavity, remove extraneously tissue, rinse with PBS, then gently blot excess fluid if weighing (liver weight is usually expressed as a percentage of total body weight). Cut a transverse section through the large left lobe and then through the two lobes including the gallbladder. Avoid over-fixation which can result in loss of glycogen (Swiss cheese morphology).
Intestinal tract: Remove from body cavity, flush out with PBS or open entire length then remove ingesta, place about 1 to 1.5 cm sections each of small intestine (SI) and large intestine (LI) on filter paper to maintain linearity. Harvest the same intestinal regions between animals. You can also roll up the entire SI or LI as a "Swiss roll".
Stomach- Open along greater curvature, rinse off ingesta, trim off upper half (squamous mucosa), make longitudinal cuts that include the entire longitudinal length including the proximal fundus, distal portion (antrum), and upper duodenum. Lay between 2 papers so the strips fix flat. Indicate to embed on edge.
Lungs: Remove lungs with trachea intact from body cavity, re-inflate with fixative (insert needle on syringe into trachea, pre-place a suture, re- inflate lungs gently with fixative; can also be done in situ then removed from animal. Remove needle and snug trachea suture tight.). Immersion fix inflated lungs overnight, then submit all or take coronal sections through the lobes.
Heart- Can be removed separately or with lungs. Take a longitudinal section that includes portions of valves, atria and ventricular walls or divide in thirds and submit all three..
Skin: Collect from least furry area. Obtain ~ 5 mm long and 1 mm wide rectangle of skin and place between filter papers or sponges so that it will fix flat.
Brain and testis: Best to immersion fix overnight before trimming and placing in cassettes. Bouin's fixative is preferred for testis.
Lesions and unknowns: Always take samples from tissues that seem out of the ordinary. Include normal appearing tissue for comparison when possible.
2. How do I fix my samples?
Fresh tissues may be rinsed with PBS if needed then immediately transferred to cassettes and immersed fixed to avoid autolysis. Intestine, pancreas, liver, and brain are particularly prone to autolysis so it is best to get into fixative right after harvesting. Other organs can be kept a short time in cold PBS. Soft tissues like brain, lung, or testis, are better trimmed after hardening in fixative.
See also Jackson lab's website for fixative information. or Vanderbilt's guide.
Formalin, 10% NBF : Fixation time ~12-24 hours, room temperature. Purchase from ThermoFisher (23-245-684). 10% NBF is a good general fixative for histopathology and is also good for most IHC assays if fixation time is kept to the minimum. Contains 4% w/v formaldehyde with phosphate buffers.
4% Paraformaldehyde (4%PF): Fixation time depends on assay but generally overnight in refrigerator. Paraformaldehyde is the anhydrous form of formaldehyde. It is best prepared fresh each time or stored in frozen aliquots. It is less harsh than 10% NBF and is preferred for many antigens in immunostaining, particularly direct GFP visualization, lymphocyte antigens, CNS, and others. Fixed samples can also be embedded as fixed frozens after cryoprotection in 18-30% sucrose as described above.
PLP: Fixation time 24-48 hours in refrigerator. Contains 2% paraformaldehyde, excellent for IHC on paraffin samples. Tissues are softer and can be somewhat difficult to section.
Bouin's: Fixation time 6 hours. Purchase from Fisher or Sigma. Sometimes preferred for histopathology for certain organs like testes and pancreas and for immunostaining with certain antibodies (islets).
3. How do I submit my samples?
Submit wet tissues in containers or trimmed in cassettes after the fixation is ended. It is much preferable to submit trimmed tissues in cassettes because then you determine the size and shape of the samples on the slides. If you submit tissue that we have to trim and/or transfer into cassettes, there are additional charges associated due to extra effort.
Do not overcrowd tissues in the cassette. Tissues that are too big will not adequately fix, infiltrate, section, or stain well later on. If staff determine that the cassettes are overcrowded, you will be contacted to fix them or they will be divided and processed according to what staff determine will provide the best result.
We have several cassettes types free for users: regular, large, and biopsy (1- and 4-chambers)
as well as mesh bags and blue sponges. To prevent small tissues from being lost
during processing, place them into biopsy cassettes or
between sponges. The paraffin processor uses vacuum to facilitate tissue
infiltration which can coax small tissues out of regular
cassettes.
Hand-label the cassette with a #2 pencil only. Processing
solvents can remove ink labels even from some "permanent"
Sharpies. Pre labeled cassettes can be requested with the Cassette Printing Request form and is free of charge to our uses. This is the preferred method as it reduces the risk of transcription errors, loss of transcription details during processing, and is easier to read. You can also come and print out your cassettes.
Keep cassettes submerged to avoid having your samples dry out. Transport
samples to D11-41 in a leak-proof container. Make sure the container is labeled
with your name, PI and what solution the samples are in. As long as you have
emailed us your submission form and labeled your container, you do not need to
print out a hardcopy of the form as we'll do that after the form is checked in.If you want your particular container returned, you must tell the Submission staff.
Cassette IDs should match what is written on the Submission form in the Sample table.
Processing/Embedding/Sectioning
1. How will my samples be processed and embedded?
We use 70% ethanol or PBS as the first station in theSakura VIP6 automatic processor rather than formalin so that all specimens are fixed according to the user. Samples are put on the processor by 4:00 pm and held until early morning when the processor begins the dehydration, clearing, and infiltration steps followed by paraffin wax cycles. We also can put your samples on the Sakura VIP150 processor which has 10% NBF as the first station if you just harvested the samples that day.
Embedding is done first thing in the morning. Samples will be embedded as placed in the cassette unless special instructions
are given. Tissue embedded down will be cut first.
Special requests for embedding:
As in cassette (default)
Cut surface down
On End or Cross section (vessels like aorta and tubular organs (GI, uterus))
On Edge (Skin, gall bladder, opened tubular organs like GI tract)
Flat
Embryo- Head down, saggital
Specify any other embedding instructions in the Special instructions section.
If you're interested in a very small area of the tissue as for a lesion,
and you've trimmed the tissue so that the lesion area is right at the surface of your
sample, it may be lost when the block is "faced" if
you have not mentioned this to the histology staff or on your submission form. Block facing is done to get into the sample deep
enough so that a representative section of the entire sample is obtained. The
first sections will normally be discarded. In such cases, it is very
important that you discuss your special embedding requirements with the
histotechnician beforehand and write this
information in the special requests area on the submission form.
You are welcome to draw a picture of what you want the final slide to
look like using the slide diagram on the submission form page 2.
2. How are my blocks sectioned?
Paraffin blocks are sectioned at 4 microns with generally a single section placed on a Superfrost
Plus slides (12-550-15, Fisher). You
can request different section thickness.
You can request multiple sections per slide; number will depend on your
block's face size.
You can also request exact serial sections where 5-6 serial sections from a ribbon are
placed on numbered slides.
You can request step levels where different levels of the block are examined.
These can be specified as surface then 2/3 way through or by specifying the
step distance (example: 50-100um steps).
Additional efforts performed to accomplish your submission may require technical charges.These are billed in 15-minute increments.
If DNA or RNA is the product of interest, indicate this under Special requests.