May 28, 2025
Histology Protocol for Paraffin Embedded Mouse Knee
- Camilla Majano1,
- Carolina Leynes1,
- Ming-Ming Jiang1,
- Yangjin Bae1,
- Nele Haelterman1,
- Blee 1
- 1Baylor Department of Human Molecular Genetics
Protocol Citation: Camilla Majano, Carolina Leynes, Ming-Ming Jiang, Yangjin Bae, Nele Haelterman, Blee 2025. Histology Protocol for Paraffin Embedded Mouse Knee. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw19qdlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 17, 2024
Last Modified: May 28, 2025
Protocol Integer ID: 98286
Keywords: histology protocol for paraffin embedded mouse knee, mouse knee joints for paraffin, paraffin embedded mouse knee, mouse knee joint, histology protocol, based histological analysis, histological analysis, paraffin
Funders Acknowledgements:
Brendan Lee
Grant ID: 1 UC2 AR082200-01
Abstract
This method describes how to harvest, embed, and process, mouse knee joints for paraffin-based histological analysis.
Materials
| Name | Vendor | Product Number | RRID | |
| Ammonium Hydroxide (NH4OH) (Certified ACS Plus), Fisher Chemical | Fisher Scientific | A669C-212 | ||
| Ethanol | ||||
| Ethylenediaminetetraacetic acid (EDTA free acid) | Amresco | 0322-1KG | ||
| Fisherbrand Tissue Path IV Tissue Cassettes | Fisher | 22-272416 | ||
| Isoflurane | ||||
| Milli Q Water | ||||
| Paraffin | ||||
| Paraformaldehyde reagent grade, crystalline | Sigma-Aldrich | P6148-500g | ||
| Phosphate-buffered saline (PBS, 10X), RNAse free | VWR | AAJ62851-AK | ||
| Sodium Hydroxide solution, 10.00N | VWR | VW3247-4 | ||
| Slide Superfrost + 25*75mm | VWR | 48311-703 | ||
| Tissue-Tek Mega-Cassette System mega base mold, metal | Electron Microscopy Sciences/ VWR | 62512-31 | ||
| Tris-HCL | Sigma | 10812846001 |
Troubleshooting
Safety warnings
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Ethics statement
Procedures involving animal subjects were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine.
Harvesting Knee Joints
Set up dissection room: dissection tools, bucket of ice, container with 1X PBS, diapers, isoflurane for euthanization, labeled glass vials or cassettes for each sample
Keep 4% PFA, 1X PBS, and sample vials/cassette container on ice
Add 4% PFA into individual labeled glass vials or into the large container that will hold cassettes.
Euthanize mouse by placing in container with isoflurane.
Collect hindlimbs:
Spray with alcohol.
Make a small incision on the abdominal area. Pull from opposite ends of the incision to remove the skin to expose one leg at a time
Cut off the exposed limb by cutting through the upper portion of the femur.
Trim muscle from hindlimb as shown in the image.
Drop-fix:
Place tissue in PFA-filled glass vials or in labeled cassette (and place cassette with sample into container). For coronal sections, fix joint in a natural position (or a straight position as shown in the figure below). For sagittal sections, fix joint in a 90 degree angle.
For cassettes, arrange knees as shown below
Preparation for harvest
Prepare the 4% Paraformaldehyde (PFA). This must be done under the fume hood. (0.04*V = g needed)
Set up materials needed inside the hood: magnet, stir bar, scale, 500mL glass beaker, sodium hydroxide (NaOH, 10N)
Calculate how much PFA solution you need. ( # of knees * 25mL = total PFA needed if using glass scintillation vial)
Microwave calculated amount of 1X PBS in a beaker (1 min / every 100mL ) (ex: 2 min for 200mL)
Cover flask with aluminum and transfer into fume hood. Add 10ul (for every 100mL) of 10N sodium hydroxide solution
Dissolve calculated amount of PFA (0.04*V = grams) (located inside 4 degree common space) with heated 1X PBS
Place magnet into solution, cover, and stir for about 15 min or until fully dissolved
Check the pH of the solution (must be between 7.4-7.6) with pH probe. Carefully move pH probe under hood to protect yourself from fumes.
If you need to bring down the pH, use HCL 1N (1M). Add small amounts until reach correct pH
Transfer solution into 500mL glass container and cool down container in ice bucket
Fixation
Once all samples are harvested, fix tissues for 48 hrs at room temperature with a gentle agitation.
Wash
After fixation, dispose of 4% PFA solution following institution guidelines.
Wash with 1X PBS for 30 mins for three times each with gentle agitation at room temperature.
Note: If samples need to be stored prior to being decalcified it can be done after fixation and washing steps (3x for 30min with 1x PBS)
Store samples in 70% EtOH in 4 degrees.
Decalcification
While washing, make 10% EDTA (This can be made ahead of time and stored for long periods of time. If using stored EDTA check the pH and adjust as needed).
For 4L of 10% EDTA, add 400g of EDTA free acid to ~2.8L of Milli Q water
Add NH4OH at room temperature with continuous mixing (use a stir bar) until dissolved
Note: will take ~240-320 ml of NH4OH to get the EDTA to completely dissolve (solution may have a slight yellowish tint, but that’s normal)
Add 400ml 10X PBS and mix
Adjust to pH 7.2-7.4 with NH4OH
Note: if needed, use glacial acetic acid instead of HCL (pH=2) to bring pH value down.
Bring volume up to 40L with Milli Q water
After fixation with 4% PFA, wash samples 3 x 30min with 1X PBS at room temperature with light
agitation
Incubate tissues in 10% EDTA for 5 days at 4 degrees with light agitation
Decant and replace with fresh 10% EDTA for an additional 5 days
Do not decalcify for more than 10 days.
Confirm decalcification by x-ray
Wash samples 3 x 30 min with 1X PBS at 4 degrees with agitation
Store samples in 70% EtOH in 4 degrees until needed
Dehydration
The day before samples are to be embedded, they need to be dehydrated.
Wash samples with 95% EtOH for 1hr.
Wash samples with 100% EtOH for 1hr. Leave samples in fresh 100% EtOH overnight at room temperature.
Next day, wash with fresh 100% EtOH for 1 hr and transfer samples to the Epredia automatic tissue processor machine.
Tissue Processing
Use the following setting for Epredia automatic tissue processor [STP 120-3], 4.5 hrs processing time [Program 1]:
3 xylene washes for 2 x 20 minutes, and 1 x 30 minutes
2 Paraffin washes for 2 x 1 hr. After being in the last paraffin wash for at least 1h, stop the program and transfer samples to Tissue-Tek embedding machine.
Frontal Paraffin Embedding
Turn on embedding machine [Sakura Tissue-Tek TEC] at least 1hr before use. Warm up large metal molds. Turn on cryo snap freeze when you are about to start embedding.
Remove mold from the holding area and fill up a large mold with melted paraffin.
Trim excess tissue that was used to fix the joint in a natural position.
Locate the patella tendon as seen below with the red arrow. Using forceps, gently hold the medial and lateral side of the knee so the posterior side faces you and the frontal side faces the bottom of the mold shown below.
Place knee frontal side down facing the base of the mold.
Hold in place while quick freeze.
Add more paraffin to the mold. Do not place mold back onto the hot surface and continue quick freezing. Place cassette on top, add more paraffin and then place on cold plate. Molds take about 30 minutes to harden.
Paraffin Coronal Plane Sectioning
Set up microtome workspace – you will need your collection slides, warm water bath @ 45C, brushes, razor, blade, microtome [Leica RM2265], hotplate, sample blocks, light microscope, paper towels, ice.
While setting up, cut off the excess of paraffin from your sample block at an angle before mounting it on the stage (see image below)
Use paint brushes to assist in keeping ribbons from rolling/folding.
While trimming at 10um - 30um, adjust the sample block until the tissue is sectioned in the correct plane (ex: frontal). You can check how deep in the joint you are by separating a ribbon with the razor and gently moving it to the water bath. Gently fish out the ribbon with a slide, gently tap the slide on a paper towel for excess water to run off, and then view under light microscope.
Trouble shoot: If ribbons begin to shred or fold too much, rub a small piece of ice on the sample block to cool down the paraffin. Keep a small plate of ice next to you for this reason
Set 7 um for collecting section. Start collecting when you see the landmarks. (Anterior landmark: the menisci (lateral/medial) is connected with ligament (dotted line) and/or the condyle begins to show (asterisk). Cut through to posterior (Landmark: the menisci (lateral or medial) reconnect with the tibia/femur)
Section collection:
Collect serial sections (3 ribbons on each slide, 7uM thick).
Collect until end of the target area. Expect between 25-40 slides, depending on how soon you begin to collect.
Staining
Before staining, make sure to bake slides horizontally in 54 degree incubator for 1 hour. This will help with tissue adherence to slides.
Hematoxylin-Eosin staining (H&E)
Make 250mL of 1% sodium bicarbonate (2.5g NaCO3 dissolved in 250mL milliQ H2O) prior to starting
Solutions and Reagents:
Mayer's Hematoxylin Solution (Sigma)
Eosin Y Solution Alcoholic (Sigma)
Deparaffinize sections, 2 changes of xylene, 3 minutes each.
Re-hydrate in 2 changes of absolute alcohol, 2 minutes each.
95% alcohol for 2 minutes and 80% alcohol for 2 minutes.
Wash briefly in distilled water.
Stain in Mayer hematoxylin solution for 3 minutes.
Rinse in distilled water.
1% NaHCO3 for 1 minute (make fresh solution daily).
Rinse with running DI water for 1 minute.
Rinse in 80% alcohol, 30 seconds.
Counterstain in eosin-Y alcoholic solution for 1 minute.
Destain in 80% alcohol for 1 minute.
Dehydrate through 95% alcohol, 2 changes of 100% alcohol, 1 minute each.
Clear in 2 changes of xylene, 2 minutes each.
Mount with xylene based mounting medium.
Safranin-O staining
Materials:
100% EtOH – need two containers for deparaffinizing samples and two for dehydrating after staining
95% EtOH – need one container for deparaffinizing samples and one for the Safranin O rinse
80% EtOH – need one container for deparaffinizing samples
Weigert’s Iron Hematoxylin Set – combine 100ml Solution A and 100ml Solution B to make working solution for staining
1% Acid-Alcohol – add 5ml of hydrochloric acid (HCL, 36.5-38.0%) and 350ml 100% EtOH to 145ml Milli Q water
0.02% Fast Green – add 0.1g of Fast Green powder to 500ml Milli Q water
1% Acetic Acid-Alcohol – add 5 ml of glacial acetic acid and 350ml 100% EtOH to 145ml Milli Q water
1% Safranin O – add 5g of Safranin O powder to 500ml Milli Q water
Xylene
Cytoseal XYL
Note: between each change of solution, dab slides while still in the stand on a paper towel to remove excess liquid
Place in xylene and incubate for 5 min
Transfer to a new container of xylene and incubate for 3 min
Transfer again to new container of xylene and incubate for 3 min
Transfer to 100% EtOH and incubate for 2 min
Repeat again to new container of 100% EtOH and incubate for 2 min
Transfer to 95% EtOH and incubate for 2 min
Transfer to 80% EtOH and incubate for 2 min
Transfer to distilled water and incubate for 0.5-1min
Place in Weigert’s Iron Hematoxylin for 5 min
Wash gently in 4 changes of distilled water
Differentiate in 1% Acid-Alcohol for 2 sec (dipping slides 3 times should be sufficient)
Rinse gently in 3 changes of distilled water
Place in 0.02% Fast Green. For 1 min (do not rinse)
Place in 1.0% Acetic Acid for 30s (do not rinse)
Place in 1.0% Safranin O for 30 min (do not rinse)
Rinse briefly in 95% EtOH (dipping slides 3 times should be sufficient)
Dehydrate with two changes of 100% EtOH and three changes of xylene (each for 1 min)
Mount using Cytoseal XYL, and allow to cure for a minimum of 24h
Expected results and controls
Successful histology will yield clear strong staining.
Red stain of Safranin-O staining indicates proteoglycans in joint tissue.
Troubleshooting
Check that 4% PFA is between 7.4 – 7.6 pH when made and before tissue harvest. If it is too acidic it will be too harsh for tissue, if too basic it will not penetrate the tissue at all.
Check that 10% EDTA is between 7.2 – 7.4 pH (preferably 7.3 pH) when made and before decalcification step. If it is too acidic it will over decalcify tissue, if too basic it will not decalcify tissue sufficiently.
During sectioning, samples can be re-embedded if cannot reach desired coronal plane with microtome stage adjustments.
Before staining process make sure reagents are fresh. Make sure to bake slides in 56 degree incubator in order to strengthen tissue section attachment to slides.