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Background: Histology assessments are useful to examine tissue elements like cellular components, organelles, and ECM structure. Histology staining can be done with multiple samples through formalin fixation and paraffin embedding, then adding stains such as hematoxylin and eosin (H&E) or Masson’s Trichrome to observe the samples through fluorescence or bright-field microscope.
Need within our aims: We performed histology assessments as a way of qualitatively assessing our success at decellularizing the porcine skeletal muscle tissue. By staining slide samples, we wanted to look for noticeable changes in tissue composition.
Experiment: Every time that we performed our decellularization protocol, we collected tissue samples on days 3, 4, and 5 of the protocol. We embedded the samples in OCT (optimal cutting temperature compound) to later be able to slice the tissue using a cryostat. After slicing the samples and preparing our slides, they were sent for staining. We chose to perform both H&E and Trichrome staining. We specifically chose to perform Trichrome staining because it is most used to look at connective tissues and collagen in tissue structures.
Our general workflow for qualitative testing of complete decellularization of the tissue:
Collected tissue sample embedded in OCT block
Cryostat sectioning of tissue into slices
Slide with tissue slices; porcine paraspinal tissue - decell day 3
Tissue sample visualization with automated microscope and Invitrogen EVOS FL Auto 2 Imaging System
Results & Interpretation: Our results can be visualized below. As can be seen from our pictures, there is progressive removal of cellular content. This qualitatively depicts successful decellularization, as the DNA and cells are being washed away while leaving the ECM and collagen structures intact.
Looking more closely at the pictures, there is a noticeable difference between Day 0 (native) and Day 3 tissues, and also between Day 4 and Day 5 tissues. By Day 5, however, there is not much distinguishable change compared with Day 4.
This led us to hypothesize and infer that a 4-day decellularization protocol was probably optimal to balance between completely decellularizing the tissue without damaging or degrading the ECM and collagen structures. Further testing to standardize this within our optimized protocol was performed through DNA quantification.
H&E Staining
Hematoxylin stains nuclei blue
Eosin stains the ECM and other structures pink
Trichrome Staining
Nuclei are stained dark, reddish brown
Muscle tissue is stained red
Collagen structures are stained blue
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