Processing of Lentivirus-Transduced Mouse Intestinal Organoids for Immunohistochemical Analysis

Streszczenie

Source: Van Lidth de Jeude, J. F., et al. A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids. J. Vis. Exp. (2015).

This video demonstrates the processing of lentivirus-transduced mouse intestinal organoids for paraffin embedding, to enable their downstream analysis for assessing gene expression.

Protokół

1. Processing organoids for paraffin embedding and immunohistochemistry

1. Prior to initiation of organoid embedding, pre-warm an aluminum block with holes fitting 12 mm diameter glass tubes in incubator at 70 °C to keep paraffin liquid.

2. Take a single well with full-grown organoids and remove the medium, leaving the embedded organoids intact.

3. Add 1 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS) straight to well and fix at 4 °C anywhere from 1 hr to overnight.

4. Replace paraformaldehyde with 1 ml of ice cold PBS.Optionally: keep fixed organoids in PBS up to 1 week at 4 °C after this step.

5. Resuspend fixed organoids in 1 ml of PBS and place into glass vial.

6. Let organoids sink to bottom for 1 min, decant PBS and replace with 70% ethanol in which a couple of droplets of eosin solution are dissolved to enable visualization of organoids throughout the embedding process.

7. Leave organoids in 70% ethanol at room temperature. After 30 min, remove 70% ethanol by carefully decanting, being able to visualize the organoids by eye because of slight pinkish eosin color. Replace embedding solution with 96% ethanol.

8. Repeat step 1.7, each time replacing the embedding solution with the next one. Pass organoids through the following solutions subsequently: 70% ethanol, 90% ethanol, 96% ethanol, 100% ethanol, 100% ethanol, xylene, xylene.

9. Decant the last xylene wash and pour paraffin into the tube. Put the tube immediately in the pre-warmed aluminum block at 70 °C for 30 min and replace the paraffin with new clean paraffin.

10. Pour paraffin off and pipette organoids into the paraffin block mold using a pre-warmed Pasteur pipette with a large opening. Keep Pasteur pipette warm using a Bunsen burner. Place all organoids in the paraffin block mold in a small layer of liquid paraffin.

11. As much as possible, try to manipulate all organoids towards the center of the paraffin block mold using a warmed dissection needle. Keep the dissection needle warm using a Bunsen burner.

12. When localization of organoids in the mold is satisfactory, chill mold slightly to solidify the paraffin layer.

13. Finish the block by pouring more paraffin on top and add a standard histological embedding cassette.

Materiały

Lista materiałów użytych w tym artykule

Nazwa	Firma	Numer katalogowy	Komentarze
Paraformaldehyde	Sigma	252549-1L
Glass vial conical 12 mm x 75 mm 5 ml	VWR	LSUKM12
Eosin Yellowish	VWR	1,159,350,025