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1. Decellularization
NOTE: This procedure was adapted from previously published methods focused on adipose decellularization, which included the sodium deoxycholate ionic detergent rather than sodium dodecyl sulfate to remove DNA efficiently.
- On day 1, remove frozen Mammary Fat pads or MFPs from -80 °C and thaw at room temperature.
- Once thawed, dry MFPs briefly on a delicate task wipe. Weigh the MFPs using an analytical scale.
- Using a pair of forceps with scissors or a scalpel, divide tissue up into 3 mm x 3 mm x 3 mm samples for study of the intact ECM and the remaining tissue for hydrogel production.
NOTE: The number of samples is dependent on the number of testing methods, e.g. the collection of two samples is described below: one for paraffin embedding (step 1.5) and one for freezing in cryostat embedding medium, if desired (see the Table of Materials and step 1.6).
- Weigh the tissues. If embedding in paraffin for sectioning, continue to step 1.5. If freezing in cryostat embedding medium for sectioning, continue to step 1.6.
- In a chemical hood, submerge the tissue in 10% neutral buffered formalin (NBF) (see the Table of Materials) for 24 h at 4 °C. Wash 3 times in PBS for 5 min each. Submerge the tissue in 30% sucrose for 48 h at 4 °C.
- Weigh the tissue now that this piece has been removed. Continue to step 1.6.
- In a chemical hood, place MFP pieces in a labeled cassette prepped with cryostat embedding medium. Add more cryostat embedding medium to cover the tissue.
- Place the cassette into a beaker of 2-methylbutane (see the Table of Materials) that is pre-cooled with liquid nitrogen. The beaker should have enough 2-methylbutane to cover the bottom but not enough to submerge the cassette because the cryostat embedding medium should not touch the 2-methylbutane. Let the cassette sit in the 2-methylbutane until the cryostat embedding medium freezes and becomes opaque.
- Wrap the cassette(s) in foil, label, and leave at -80 °C until used for sectioning.
NOTE: Tissues placed immediately in cryostat embedding medium were sectioned at 5 μm while tissues incubated in sucrose were sectioned at 30 μm to retain adipocyte morphology.
- Use forceps to manually massage the remaining tissue.
NOTE: Tissue pieces may also be placed in 10% NBF for 24–48 h, rinsed in PBS, and left in 70% ethanol until embedding in paraffin. Following embedding, 5 μm sections can be used for hematoxylin and eosin (H & E) staining (see section 2 below).
- Place the MFPs in 6 cm dishes with 5 mL 0.02% trypsin/0.05% EDTA solution. Incubate at 37 °C for 1 h. Spray and wipe the dishes with 70% ethanol before placing in the incubator.
- Use 0.7 mm strainers to wash the MFPs with deionized (DI) water by pouring water over the tissue three times. Use forceps to manually massage the tissue in between washes.
- Briefly dry tissue on a delicate task wipe and weigh. Place tissues in a pre-autoclaved beaker containing an appropriately sized stir bar. Cover tissues with 60 mL of 3% t-octylphenoxypolyethoxyethanol (see the Table of Materials) per 1 g of tissue and stir for 1 h at room temperature. Use a minimum of 20 mL.
- Dump tissue and contents into a strainer. Rinse the beaker with DI water and pour onto tissues. Repeat two more times. Use forceps to manually massage the tissue in between rinses.
- Briefly dry tissue on a delicate task wipe and weigh. Place tissues and stir bars back in the same beakers, and cover with 60 mL of 4% deoxycholic acid per 1 g of tissue. Stir for 1 h at room temperature. Use a minimum of 20 mL.
- Dump tissue and contents into a mesh strainer. Rinse the beaker with DI water and pour onto tissues. Repeat two more times. Use forceps to manually massage the tissue in between rinses.
- Briefly dry tissue on a delicate task wipe and weigh.
- Place tissues in the same beaker with fresh DI water supplemented with 1% penicillin-streptomycin. Cover tightly with paraffin film. Leave overnight at 4 °C.
- Wash strainers and beakers for use the following day.
- On day 2, drain beaker contents into a strainer. Briefly dry tissue on a delicate task wipe and weigh.
- Place MFPs in the same beaker with an appropriately sized stir bar. Cover with 60 mL 4% ethanol/0.1% peracetic acid solution per 1 g of tissue. Use a minimum of 20 mL. Stir for 2 h at room temperature.
- Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue. Place contents back into the beaker. Wash tissue by covering it with 60 mL of 1x PBS per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat once.
- Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue. Place contents back into beaker. Wash tissue by covering it with 60 mL DI water per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat once.
- Briefly dry tissue on a delicate task wipe and weigh. Dump tissue and contents into a strainer. Use forceps to manually massage the tissue.
- Place contents back into beaker. Cover tissues with 60 mL of 100% n-propanol per 1 g of tissue. Use a minimum of 20 mL. Stir for 1 h at room temperature.
- Briefly dry tissue on a delicate task wipe and weigh. Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue.
- Place contents back into beaker. Wash tissue by covering it with 60 mL of DI water per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat three times.
- Dump tissue and contents into a strainer. Repeat steps 2.3–2.6 to collect pieces of tissue for sucrose incubation and freezing in cryostat embedding medium.
- Briefly dry tissue on a delicate task wipe and weigh. Place in a labeled 15 mL tube. Freeze at -80 °C overnight.