Aug 13, 2025
Knee histology protocol for immunohistochemistry on cryosections
- Carolina Leynes1,
- Zelong Dou1,
- Camilla Majano1,
- Ming-Ming Jiang1,
- Matthew Grol2,
- Nele Haelterman1,
- Yangjin Bae1,
- Brendan Lee1
- 1Baylor Department of Human Molecular Genetics;
- 2University of Western Ontario Schulich School of Medicine & Dentistry
- RE-JOIN
Protocol Citation: Carolina Leynes, Zelong Dou, Camilla Majano, Ming-Ming Jiang, Matthew Grol, Nele Haelterman, Yangjin Bae, Brendan Lee 2025. Knee histology protocol for immunohistochemistry on cryosections. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjny7bgk5/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 18, 2024
Last Modified: August 13, 2025
Protocol Integer ID: 98439
Keywords: immunohistochemical analysis of mouse knee joint, knee histology protocol for immunohistochemistry, cryosections osteoarthritis, mouse knee joint, knee histology protocol, immunohistochemical analysis, immunohistochemistry, joint biology, joint capsule, cartilage, assessment of neural innervation, knee, continued investigation into knee, neural innervation, ligament, joint involvement
Funders Acknowledgements:
Department of Defense (DOD), Combinatorial helper-dependent adenoviral gene therapy for post-traumatic osteoarthritis
Grant ID: PR210221
Neuronal anatomy, connectivity, and phenotypic innervation of the knee joint
Grant ID: UC2 AR082200-01
Abstract
Osteoarthritis (OA) is a chronic, degenerative condition characterized by whole-joint involvement, affecting the bone, cartilage, synovium, ligaments, and joint capsule. Given its widespread impact on millions of individuals, continued investigation into knee joint biology and pathology is critical. Here, we present a detailed protocol for the dissection, processing, cryosectioning, and immunohistochemical analysis of mouse knee joints. This method enables high-quality preservation of joint architecture and is optimized for the assessment of neural innervation using anti–βIII-tubulin, a pan-neuronal marker.
Materials
1. Materials for collecting, processing, and sectioning knee joints:
| A | B | C | D | |
| Name | Vendor | Product Number | RRID | |
| Ammonium Hydroxide (NH4OH) (28-30%) (Certified ACS Plus), Fisher Chemical | Fisher Scientific | A669C-212 | ||
| CryoTape – Tissue Tape Window | Fisher Scientific | 50-303-81 | ||
| CryoJane Adhesive Coated Slides | Fisher Scientific | 50-304-08 | ||
| Ethanol | ||||
| Ethylenediaminetetraacetic acid (EDTA free acid) | VWR | 0322-1KG | ||
| Isoflurane | ||||
| Milli Q Water | ||||
| Paraformaldehyde(PFA) reagent grade, crystalline | Sigma-Aldrich | P6148-500g | ||
| Phosphate-buffered saline (PBS, 10X), RNAse free | VWR | AAJ62851-AK | ||
| Sodium Hydroxide solution, 10N | VWR | VW3247-4 | ||
| Sucrose, ACS, EMD Millipore | Sigma-Aldrich | SX1075-7 | ||
| Tissue-Tek - 25 x 20 x 5 mm Cryomold | Tissue-Tek | 25608-916 | ||
| Tris-HCL | Sigma | 10812846001 | ||
| 20 mL Glass Vials | VWR | 89093-854 | ||
| Optimal cutting temperature (OCT) media | VWR | 25608-930 | ||
| Acetic acid glacial solution, (>80 percent acid, by mass) | ||||
| Leica CM3050 S Cryostat | Leica | |||
| Petri Dish (steril) for Bact. Size 100 x 15 mm | VWR | 25384-302 |
2. Materials needed for IHC on knee sections
| A | B | C | D | |
| Name | Vendor | Product Number | RRID | |
| Pap-pen | FisherScientific | NC9827128 | ||
| Phosphate-buffered saline (PBS, 10X), RNAse free | VWR | AAJ62851-AK | ||
| Normal Donkey Serum (NDS) | VWR | 017-000-121 | ||
| Tween R 20 (Polysorbate) | VWR | 97063-872 | ||
| GFP Polyclonal antibody | Invitrogen | A-6455 | AB_221570 | |
| Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexas Fluor 647 | Invitrogen | A-31573 | AB_2536183 | |
| Living Colors DsRed Polyclonal antibody | Takara | 632496 | ||
| Anti-beta III Tubulin antibody - Neuronal Marker | Abcam | ab18207 | AB_444319 | |
| Collagen II Monoclonal Antibody | ThermoFischer Scientific | M2139 | ||
| Milli Q Water | ||||
| ProLong‱ Glass Antifade Mountant | Thermo Fisher | P36980 | ||
| Ihc World LLC IHC TEK DAPI SOLUTION 50ML | FisherScientific | IW-1404 | ||
| THORLABS INC Precision Cover Glasses, #1.5H Thickness, 22 x 22 mm, Pack of 1000 | FisherScientific | NC1415511 | ||
| Proteinase K, Recominant, PCR Grade | Sigma | 3115828001 | ||
| Dako Target Retrieval Solution, Citrate pH 6 (x 10), Concentrate, Immunohistochemistry , 500 mL | ||||
| Non-Sterile Slide-Fix™ PAP Jars | Evergreen | 240-5420-G8K |
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.
Reagent Preparation
Prepare 4% Paraformaldehyde (PFA) dissolved in 1x phosphate buffered saline (PBS)
Note: should be made fresh; max storage time is 1 week at 4℃. Make sure to prepare this reagent in the chemical hood.
Calculate grams(g) of PFA needed (0.04*Volume = g needed).
Note: If using glass vials (Refer to Materials #15), the volume needed is 20mL per vial.
Microwave 1X PBS in a beaker. For every 100 mL heat for 1 min.
Example: 2 min for 200 mL
Cover the glass beaker with aluminum foil
Transfer warm 1XPBS to the chemical hood and add 10 µL of 10N sodium hydroxide for every 100 mL
Example: 20 µL of sodium hydroxide for 200 mL of 1XPBS
Add PFA and stir bar to the heated PBS. Cover with aluminum and stir for ~ 15min or until fully dissolved
Check the pH of the solution (must be between 7.4-7.6) with pH strips
Transfer the solution into a glass bottle/container and store at 4℃ for up to 1 week
Prepare 14 % Ethylenediaminetetraacetic acid (EDTA).
Note: This solution can be stored for months, does not have to be made fresh. Make sure to prepare this reagent in the chemical hood.
To prepare 1 L, dissolve 140g EDTA free acid in 800 mL of Milli Q Water (MW)
Add 90 mL of ammonium hydroxide solution and the magnet into the beaker containing EDTA and water. Stir until fully dissolved. If it does not dissolve within 15 min of stirring add more ammonium hydroxide until EDTA dissolves
Bring volume to 1L and check that the pH falls within 7.2-7.4
If the pH is above 7.4, use acetic acid glacial to lower the pH
Transfer the solution into a glass bottle/container and place in 4℃ fridge indefinitely.
Make sure to check the pH prior to using it. Adjustment of the pH is essential.
If the pH is too low, it works only as a too-weak acid.
If the pH is too high (above 8), decalcification is accelerated but alkalinity
can be damaging to tissues
Prepare fresh 30% sucrose dissolved in 1XPBS
Note: should be prepared on the day of the experiment
Calculate grams(g) of sucrose needed (0.3*Volume = g needed).
Note: If using glass vials (Refer to Materials #15), the volume needed is 20mL per vial.
Add sucrose into a beaker containing 1XPBS. The volume of 1XPBS should be 20% less than the volume needed to adjust for the addition of the sucrose.
Stir until dissolved. Add 1XPBS to match the volume used for calculations.
Transfer the solution into a glass bottle/container and place in 4℃.
Knee Joint Harvest and Fixation
Set up dissection room: dissection tools, a bucket of ice, 1X PBS, diapers, isoflurane for euthanization, labeled glass vials filled ¾ with 4% PFA, and petri dish. Keep PFA vials and PBS cold throughout the collection.
After euthanizing mice with isoflurane, spray the mouse with 70% EtOH
Make a small incision on the abdominal area. Pull from opposite ends of the incision to remove the skin exposing one leg at a time.
Once the hindlimb has been exposed, maintain the tissue moist by performing the collections over a petri dish containing cold 1XPBS.
Isolate the exposed limb by transecting the proximal portion of the femur.
Trim muscle from the hindleg by following black dashed lines in Figure 1. After trimming the muscle, trim the joint by cutting half of the femur and half of the tibia as shown by the red lines in the after-trimming image in below figure.
Figure 1. Adult knee joint before (left) and after (right) trimming excess muscle.
Fix samples by dropping them into the vials containing PFA. Ensure the knee is in a natural, slightly bent position, as shown in Figure 1.
If using a cassette for holding samples. Place the sample against a corner of the cassette with the knee joint facing up. See Figure 2.
Figure 2. Demonstration of adult knee positioning in cassette.
Fixation is done for 24 hours at 4 ℃
Knee Joint Decalcification
Dispose of 4%PFA solution following institution guidelines
Wash with 1X PBS 3 x for 30 mins each with gentle agitation at room temp.
Note: If samples need to be stored prior to being decalcified, store them in fresh 1XPBS at 4℃.
Before starting decalcification step, check the pH of your 14% EDTA solution and adjust as needed
Remove 1XPBS from the last wash and start decalcification by adding 20mL of 14% EDTA to each vial (Refer to Materials #15).
Decalcify for 3 days at 4℃ with gentle agitation.
Important: Do Not Exceed this time as fluorophores will be damaged
Knee Joint Embedding in OCT (Coronal Orientation)
After decalcifying, wash samples with 1X PBS 3 x for 30 mins each with gentle agitation at room temp
While washing, prepare 30% sucrose (1XPBS).
After the last wash, dispose of the 1XPBS and add 30% sucrose. Samples will begin to float as shown in Figure 3.
Figure 3. Knee joint (labeled by the red arrow) incubation in 30% sucrose in 1xPBS.
Place samples in 30% sucrose (1X PBS) for 24 hrs at 4℃.
Samples can be incubated in 30% sucrose longer, but it should not exceed 2 weeks.
Samples are ready for embedding once the samples sink to the bottom.
Using fine forceps, gently grab the knee joint by the sides as shown in Figure 4.
Figure 4. Knee joint after decalcifying. Forceps will be used to grab the sides (labeled by the black arrow) surrounding the patella (labeled by the red arrow).
Place tissue in the mold filled with OCT with the patella tendon facing down and flat
against the surface.
Freeze tissue mold by placing it on a flat dry ice surface. Avoid using liquid nitrogen, as it may cause cracking or uneven freezing of OCT blocks.
Note: the color of OCT will change from clear to a white color
Once all samples have been embedded, store them at -80℃ and wrap them with aluminum foil to preserve samples for months/years.
Knee Joint Coronal Cryosections
Set the temperature on Leica CM3050 S Cryostat to -20 ℃.
Place the samples, roller, paintbrush, Tissue Tape Window, blade, UV light, and
Cryojane slides into the Cryostat and let them adjust to the temperature for 20min.
After 20 min, use a Kim wipe, dry off the Leica CM3050 S Cryostat sample disk.
Apply Optimal cutting temperature (OCT) media to the Leica CM3050 S Cryostat sample disk following the circular pattern until just before the outermost ring of the sample disk is reached as shown in Figure 5.
Figure 5. Preparations for the sample disk in order to attach the OCT sample block and begin sectioning.
Place your sample on the covered disk, ensuring OCT spreads over the sample's surface.
Place the disk with the sample into the disk holder and apply a weight.
Let it sit for at least 30min inside the cryostat.
Remove the mold(do not throw it away) from the sample and load your sample for sectioning
Adjust the orientation of the sample block so that the patella tendon is even as indicated in Figure 6.
Figure 6. Diagram representing alignment of the patella when sectioning the OCT block sample. The brown portion of the diagram indicates the soft tissue surrounding the patella tendon.
Once the patella tendon is aligned as shown in Figure 6 begin trimming, this can be done at 14 um intervals.
Begin collecting 25 µm serial sections once the central region of the knee is reached. To help with section quality, use the Tissue Tape Window and CryoJane Adhesive Slides following manufacture protocol.
Note: Avoid overcrowding slides. If using THORLABS INC Precision Cover Glasses, #1.5H Thickness, 22 x22 mm, only put 2 sections per slide. Collect serial sections. The number of slides to be collected depends on the experiment. To collect the entire middle sections expect 8-10 slides with 2 sections each.
Landmarks: point when menisci thin out and disconnects from the ligament as shown in Figure 7.
Figure 7. Middle portion of the knee indicated by the disappearance of the ligament connecting the menisci to the tibia.
Collect sections until you reach the posterior side of the knee
Landmarks: point when menisci reconnect to ligament as shown in Figure 8.
Figure 8. Posterior portion of the knee as indicated by the menisci connecting to the tibia via the labeled ligament.
Store slides at -20 deg C, until needed.
DsRed and GFP Staining
Dry frozen sections on the bench for at least 30 minutes (this is to slowly
equilibrate the tissue to room temperature and to enhance the section's
adhesion to the slides). Thaw one extra section to stain with only a secondary
antibody as negative control. Whenever possible, thaw a section from a tissue
known to express the antigen analyzed. This section will be stained exactly
like the test sections and will serve as the positive control.
Draw a circle around the block contour with the pap-pen. This produces
hydrophobicity around the tissue and avoids solution dispersion during
incubation
Add 250 µL 1XPBS for 10 minutes to hydrate the section
Perform an antigen retrieval step using proteinase K. While hydrating, dilute the proteinase K with 1XPBS to 100 µg/mL.
Note: This applies if all conditions up until this point have been followed. If there were any changes in section thickness/fixation/decal time, then titration experiments may be necessary for optimal enzyme concentration.
Discard the 1XPBS. Add 150 µL of 100 µg/mL proteinase K into two of the three sections
(one section will be the negative control when possible). Leave it for 3 min.
Do not exceed that time as the tissue will begin detaching from the slide.
After 3 minutes, discard the proteinase k and begin washing the sample with 1XPBS 3
times for 5 min.
Block with 150 µL of 10% NDS and 0.2% Tween-20(in 1XPBS) for 1hr
5min before blocking time ends, dilute antibodies using 1XPBS with 10% NDS
a. GFP antibody: 1:1000
b. DsRed antibody: 1:200
Discard the blocking solution after 1hr. Wet paper towels and place them in the slide
box beneath slide holders to keep the chamber humid
Add 150 µL of diluted primary antibody to each sample. Store samples overnight (24hrs) at 4℃ overnight in a humidified incubation chamber. (cold room rocking rack)
Note: add a wet paper towel inside the slide box to avoid slides drying out.
Wash with 150 µL of 1XPBS with 0.2% tween 20 (more stringent) x3 for 15 minutes
Apply a secondary antibody conjugated to a fluorophore diluted (1:1000) in the blocking solution (10% NDS and 0.2% Tween-20(in 1XPBS)) for 1 hour
NOTE: avoid Alexa 488 and any green conjugate - it will be hard to distinguish from
bone marrow autofluorescence, especially if the appearance of the staining is
not known in advance
Wash with 1XPBS 0.2% Tween (more stringent) x3 for 15 minutes
Drop 1 drop of DAPI dye into each section and incubate in dark slide chamber for 30 min
Wash with 1XPBS 0.2% Tween (more stringent) x3 for 5 minutes
Add one drop of ProLong‱ Gold Antifade Mountant to the section; using the thin
forceps, slowly slide the coverslip on top of the section avoiding bubble
formation
Allow mounted sections to set in the dark for 18-20hrs before observing them with the microscope. See Figure 9 for expected results.
Figure 9. Expected results for GFP and DsRed IHC staining on coronal frozen sections from WT mouse injected with AAV 2/9 tdtomato + HdAdv GFP.
IHC for Tuj1 on Frozen Sections
Dry frozen sections on the bench for at least 30 minutes (this is to slowly
equilibrate the tissue to room temperature and to enhance the section's
adhesion to the slides). Thaw one extra section to stain with only a secondary
antibody as negative control. Whenever possible, thaw a section from a tissue
known to express the antigen analyzed. This section will be stained exactly
like the test sections and will serve as the positive control.
Fix Slides in 4%PFA for 30 min
Wash slides 3x with 1xPBS for 5min
Prepare a 1:10 dilution (MilliQ water) of Dako target retrieval solution
Note: For a non-sterile slide-fix Pap Jar (Catalog# 240-5420-G8K), a volume of 25 mL is needed to submerge section.
After the last PBS wash, insert slides into the Pap Jar, cap, and incubate in water bath at 60℃ overnight in appropriate rack holder
Discard solution from slide and block with 5%NDS in 0.2% Tween-20 in 1xPBS overnight.
Discard blocking solution and add diluted primary ab overnight(24hrs) at 4℃ overnight in a humidified incubation chamber. (cold room rocking rack) (+ wet paper towel in slide box)
1:200 dilution of primary ab in 5% NDS and 0.2% Tween-20(in 1XPBS)
Wash with 100 µL of 1XPBS with 0.2% tween 20 (more stringent) x3 for 15 minutes.
Apply a secondary antibody conjugated to a fluorophore diluted (1:700) in the blocking solution (5% NDS and 0.2% Tween-20(in 1XPBS)) for 1 hr.
NOTE: avoid Alexa 488 and any green conjugate - it will be hard to distinguish from bone marrow autofluorescence, especially if the appearance of the staining is not known in advance.
Wash with 1XPBS 0.2% Tween (more stringent) x3 for 15 minutes.
Dry excess water by blotting the edge of the slide against a paper towel.
Drop 1 drop of DAPI dye into each section and incubate for 30min at room temperature
Wash 3x with 1xPBS for 5 minutes
Drop one drop of ProLong™ Gold Antifade Mountant with DNA Stain DAPI on the section; using the thin forceps, slowly slide the coverslip on top of the section avoiding bubble formation.
Allow mounted sections to set in the dark for 18-20hrs before observing them with the microscope.
Note: Image as soon as possible as signal will weaken over time. Refer to Figure 10 for expected results.
Figure 10. Expected results for Tuj1 staining on frozen coronal sections. Red dashed region indicates the lateral synovium region.