TITLE: Fixation, Immunohistochemistry and in situ Hybridization of Human Lung Organoids 
AUTHORS: Morris Baumgardt, Maren Hülsemann,, Katharina Hellwig, Alina Langenhagen, Anne Voss, Simon Dökel, Achim D. Gruber, Stefan Hippenstiel, Andreas C. Hocke, Katja Hönzke

[DESCRIPTION]
This protocol describes the fixation of infected human alveolar-like organoids, as well as the sample preparation for immunohistochemistry and immunohistology of infected tissue from alveolar-like organoids. 

[GUIDELINES]
This protocol describes the processing of human alveolar-like organoids which have been grown according to Youk et al., 2020. https://doi.org/10.1016/j.stem.2020.10.004.

[STEPS]
SECTION: Fixation
1. Remove the organoid medium from the organoid containing well. Organoids should be collected from minimum two wells of a 24-well plate, to obtain a sufficient amount of organoids for staining.
SECTION: Fixation
2. Add 1 mL cold base medium and collect Cultrex with organoids in a tube, flush well with 1 mL base medium.

SECTION: Fixation
3. Place tubes with organoids at 4 °C for 5 min (to dissolve Cultrex).

SECTION: Fixation
4. Centrifuge for 300 x g, 5 min, 4 °C.

SECTION: Fixation
5. Remove supernatant, add 4% Formaldehyde solution (FA) and pipette up and down.
SECTION: Fixation
6. Store over night at 4 °C in the BSL3 lab.

SECTION: Fixation
7. Export vessel from the BSL3 with organoid and FA, centrifuge for 300 x g, 5 min, 4 °C remove FA and add fresh 4% FA.

SECTION: Fixation
8. Store over night at 4 °C.

SECTION: Fixation
9. Centrifuge for 300 x g, 5 min and remove FA.

SECTION: Fixation
10. Resolve pellet in PBS and store at 4 °C or proceed with embedding.

SECTION: Fixation
11. Centrifuge for 300 x g, 5 min and remove PBS.

SECTION: Fixation
12. Resolve pellet in 100 µL tissue Tek embedding medium (Histogel), pipette a drop on parafilm, place on ice and upon solidification place in the embedding cassette (act fast).

SECTION: Fixation
13. Close cassette, store in PBS, proceed with paraffin embedding and sectioning (sections: 2 µm).
SECTION: Paraffin embedding and sectioning
14. Put cassette into the automatic embedding machine and start the following program:


 

SECTION: Paraffin embedding and sectioning
15. In the following, after embedding, the samples are sectioned and mounted on adhesive slides. Sections of 2 µm each are prepared. Sections 1,5,9,13 are stained HE (following table) to see in which stages organoids are present, the rest is kept as blank section. 


 

SECTION: Immunohistology staining
16. Dewaxing over night at 60 °C in Roticlear.

SECTION: Immunohistology staining
17. Put the slide with organoids three times for 15 min in Roticlear at 60 °C (use fresh Roticlear for every step).

SECTION: Immunohistology staining
18. 15 min 100% Ethanol.

SECTION: Immunohistology staining
19. 1 min 100% Ethanol.

SECTION: Immunohistology staining
20. 10 min 96% Ethanol.

SECTION: Immunohistology staining
21. 5 min 80% Ethanol.

SECTION: Immunohistology staining
22. 5 min 70% Ethanol.

SECTION: Immunohistology staining
23. 5 min 50% Ethanol.

SECTION: Immunohistology staining
24. Shake 3x 5 min in 0.01 Molarity (M) PBS (100 U/min).

SECTION: Immunohistology staining
25. Antigen-Retrival for 30 min in a steam bath: Tris-EDTA buffer.

SECTION: Immunohistology staining
26. Let cool down for approx. 30 min.

SECTION: Immunohistology staining
27. Shake 3x 5 min in 0.01 Molarity (M) PBS (100 U/min).

SECTION: Immunohistology staining
28. 15 min permeabilisation in 1% Triton (in 0.01 Molarity (M) PBS) (on shaker 100 U/min).

SECTION: Immunohistology staining
29. Shake 3x 5 min in 0.01 Molarity (M) PBS (100 U/min).

SECTION: Immunohistology staining
30. Blocking for 30 min in 5% appropriate serum (in dilution medium), ca. 70 µL per tissue slice.

SECTION: Immunohistology staining
31. Flush carefully with 0.01 Molarity (M) PBS (Pasteur pipette).

SECTION: Immunohistology staining
32. Dilute primary antibody in dilution medium and incubate over night at 4 °C (wet chamber).

SECTION: Immunohistology staining
33. Flush carefully with 0.01 Molarity (M) PBS (Pasteur pipette).

SECTION: Immunohistology staining
34. Shake 3x 5 min in 0.01 Molarity (M) PBS (100 U/min).

SECTION: Immunohistology staining
35. Dilute secondary antibody in dilution medium and incubate over night at 4 °C (wet chamber).

SECTION: Immunohistology staining
36. Flush carefully with 0.01 Molarity (M) PBS (Pasteur pipette).

SECTION: Immunohistology staining
37. Shake 3x 5 min in 0.01 Molarity (M) PBS (100 U/min).

SECTION: Immunohistology staining
38. Incubate 5 min with DAPI.

SECTION: Immunohistology staining
39. Shake 3x 5 min in 0.01 Molarity (M) PBS (100 U/min).

SECTION: Immunohistology staining
40. Embedding with Mowiol® and coverslip, let air dry for 10 min and seal edges with nail polish.

SECTION: Immunohistology staining
41. Immunofluorescence is analyzed by spectral confocal microscopy using a LSM 780 [Carl-Zeiss, Jena, Germany]. Based on a spectral image lambda stack, linear unmixing of tissue autofluorescence and overlapping spectra of fluorochromes is performed using ZEN 2012 software (Carl-Zeiss, Jena, Germany). To reveal lung and cell morphology, images are combined with Differential Interference Contrast (DIC). All image sets are acquired using optimal configuration regarding resolution and signal to noise ratio. Images are processed using ZEN 2012.

   

SECTION: Viral in situ hybridization
42. We used a maximum of 12 slides at a time for the procedure. The size of the FFPE section should not exceed 2/3 of the slide and at least 5 mm away from each slide boarder.
SECTION: Viral in situ hybridization
43. Baking:
SECTION: Viral in situ hybridization
43.1. Bake the slides for 60 min at 60 °C in a dry oven.

SECTION: Viral in situ hybridization
43.2. Direct after baking remove paraffin with a tissue as much as you can, but do not touch the tissue.
SECTION: Viral in situ hybridization
44. Deparaffinization:
SECTION: Viral in situ hybridization
44.1. Pour 200 mL of xylene into a staining dish.

SECTION: Viral in situ hybridization
44.2. Transfer the baked slides to the staining dish and incubate the slides at room temperature for 5 min. Agitate frequently by moving the rack up and down.

SECTION: Viral in situ hybridization
44.3. Repeat the xylene washing with fresh xylene for 2 times.
SECTION: Viral in situ hybridization
44.4. Remove the slides from the xylene and wash the slides twice with 200 mL of 100% ethanol for 5 minwith frequent agitation.

SECTION: Viral in situ hybridization
44.5. Remove the slides from the ethanol and place them face up on a paper towel to air dry for 5 min at room temperature.

SECTION: Viral in situ hybridization
45. Hydrophobic barrier:
SECTION: Viral in situ hybridization
45.1. Before using the hydrophobic barrier pen dap it on a paper towel several times to ensure proper flow of the hydrophobic solution.
SECTION: Viral in situ hybridization
45.2. To create a hydrophobic barrier draw a rectangle around the organoid section, repeat it 2-4 times to ensure a solid seal.
SECTION: Viral in situ hybridization
45.3. Let the barrier dry for ~25 min (until it is really dry).

SECTION: Viral in situ hybridization
46. Heat Pretreatment:
SECTION: Viral in situ hybridization
46.1. Heat the 1x Pretreatment Solution to 90 °C in a porcelain staining jar (do not boil the Solution).

SECTION: Viral in situ hybridization
46.2. Incubate the slides for 10 min at 90 °C in the 1x Pretreatment Solution (time depends on tissue sample).

SECTION: Viral in situ hybridization
46.3. Remove the slide from the jar and submerge it directly into a clear staining dish containing 200 mL of full desalted autoclaved water (VE water), at this point the slides should not dry out.

SECTION: Viral in situ hybridization
46.4. Wash for 1 min with frequent agitation.

SECTION: Viral in situ hybridization
46.5. Repeat the wash step 46.3 and 46.4 one more time with 200 mL of fresh VE water.

SECTION: Viral in situ hybridization
46.6. Transfer the slides to a clear staining dish containing 0.01 Molarity (M) PBS.

SECTION: Viral in situ hybridization
47. Protease digestion and fixation:
SECTION: Viral in situ hybridization
47.1. Dilute the Protease (all in ViewRNA Tissue Kit) 1:100 in pre warmed 1X PBS (40 °C) and briefly vortex to mix.

SECTION: Viral in situ hybridization
47.2. Remove each slide out of the dish and flick it to remove excess PBS. Do not let the slides dry out.
SECTION: Viral in situ hybridization
47.3. Place each slide face up on an object carrier box (OCB; pre tempered to 40 °C / pre wetted with VE water) and immediately add 200 µL of the working protease solution onto the tissue section. Make sure that the tissue section is covered with working protease solution.

SECTION: Viral in situ hybridization
47.4. Incubate the OCB in an isotemp oven (Thermo Fisher) at 40 °C for 20 min (Time depends on organoid sample).

SECTION: Viral in situ hybridization
47.5. After incubation decant the working protease solution from the slides and wash the slides two times with PBS in a staining dish for 1 min.

SECTION: Viral in situ hybridization
47.6. Remove each slide out of the dish and flick it to remove excess PBS. Do not let the slides dry out.
SECTION: Viral in situ hybridization
47.7. Place each slide on a paper towel under a fume hood and incubate with 4% PFA for 5 min at room temperature.

SECTION: Viral in situ hybridization
47.8. After incubation decant the PFA from the slides and wash the slides two times with PBS in a staining dish for 1 min.

SECTION: Viral in situ hybridization
48. Hybridization:
SECTION: Viral in situ hybridization
48.1. Dilute the viewRNA probe set 1:40 in pre warmed probe set diluent and briefly vortex to mix. For positive control we use a EF1α probe set (also possible is ACTB or GAPDH) and for negative control we use a DapB probe set.
SECTION: Viral in situ hybridization
48.2. Remove each slide out of the dish and flick it to remove excess PBS. Do not let the slides dry out.
SECTION: Viral in situ hybridization
48.3. Place each slide face up on an OCB and immediately add 200 µL of the diluted probe set solution onto the tissue section. Make sure that the tissue section is covered with solution.

SECTION: Viral in situ hybridization
48.4. Incubate the OCB in an isotemp oven at 40 °C for 120 min.

SECTION: Viral in situ hybridization
48.5. Prepare 2 L wash buffer with VE water, 18 mL wash comp1 and 5 mL wash comp2.

SECTION: Viral in situ hybridization
48.6. After incubation decant the probe set solution and wash the slides in a staining dish with wash buffer for 2 min. Repeat washing for 2 times more with fresh washing buffer.

SECTION: Viral in situ hybridization
48.7. Optionally the slides can be stored at this point in 200 mL storage buffer (60 mL comp2 and 140 mL VE water) for 1440 min at RT.

SECTION: Viral in situ hybridization
48.8. After storage the slides must be washed again 3 times with wash buffer.
SECTION: Viral in situ hybridization
49. Pre amplifier hybridization:
SECTION: Viral in situ hybridization
49.1. Pre warm the pre amplifier mix and swirl it to mix the solution.
SECTION: Viral in situ hybridization
49.2. Remove each slide out of the dish and flick it to remove excess liquid. Do not let the slides dry out.
SECTION: Viral in situ hybridization
49.3. Place each slide face up on an OCB and immediately add200 µL of the pre amplifier mix onto the tissue section. Make sure that the tissue section is covered with solution.

SECTION: Viral in situ hybridization
49.4. Incubate the OCB in an isotemp oven at 40 °C for 25 min.

SECTION: Viral in situ hybridization
49.5. After incubation decant the pre amplifier mix and wash the slides in a staining dish with wash buffer for 2 min. Repeat washing for 2 times more with fresh washing buffer.

SECTION: Viral in situ hybridization
50. Amplifier hybridization:
SECTION: Viral in situ hybridization
50.1. Pre warm the amplifier mix and swirl it to mix the solution.
SECTION: Viral in situ hybridization
50.2. Remove each slide out of the dish and flick it to remove excess liquid. Do not let the slides dry out.
SECTION: Viral in situ hybridization
50.3. Place each slide face up on an OCB and immediately add 200 µL of the amplifier mix onto the tissue section. Make sure that the tissue section is covered with solution.

SECTION: Viral in situ hybridization
50.4. Incubate the OCB in an isotemp oven at 40 °C for 15 min

SECTION: Viral in situ hybridization
50.5. After incubation decant the amplifier mix and wash the slides in a staining dish with wash buffer for 2 min. Repeat washing for 2 times more with fresh washing buffer.

SECTION: Viral in situ hybridization
51. Label Probe 1-AP hybridization:
SECTION: Viral in situ hybridization
51.1. After incubation decant the amplifier mix and wash the slides in a staining dish with wash buffer for 2 min. 

SECTION: Viral in situ hybridization
51.2. Repeat washing for 2 times more with fresh washing buffer.
SECTION: Viral in situ hybridization
51.3. Remove each slide out of the dish and flick it to remove excess liquid. Do not let the slides dry out.
SECTION: Viral in situ hybridization
51.4. Place each slide face up on an OCB and immediately add 200 µL of the label probe 1-AP solution onto the tissue section. Make sure that the tissue section is covered with solution.

SECTION: Viral in situ hybridization
51.5. Incubate the OCB in an isotemp oven at 40 °C for 15 min.

SECTION: Viral in situ hybridization
51.6. After incubation decant the amplifier mix and wash the slides in a staining dish with wash buffer for 3 min. 

SECTION: Viral in situ hybridization
51.7. Repeat washing for 2 times more with fresh washing buffer.
SECTION: Viral in situ hybridization
52. Fast Red Substrate:
SECTION: Viral in situ hybridization
52.1. Remove the slides from the dish and place them face up on a paper towel, immediately add 200 µL of the AP Enhancer Solution to each slide and incubate for 5 min.

SECTION: Viral in situ hybridization
52.2. Decant the AP Enhancer Solution after incubation, place each slide face up on an OCB and add directly 200 µL of fresh prepared fast red mix (for 2.5 mL fast red buffer add 40 µL fast red substrate1, vortex then add 40 µL substrate2, vortex at least add 40 µL substrate3, vortex).

SECTION: Viral in situ hybridization
52.3. Incubate the OCB at RT in the dark for 60 min.

SECTION: Viral in situ hybridization
52.4. After incubation decant the fast red mix and wash the slides in a staining dish with PBS for 1 min. 
SECTION: Viral in situ hybridization
52.5. Repeat washing for one time more with fresh PBS.

SECTION: Viral in situ hybridization
53. Counter staining:
SECTION: Viral in situ hybridization
53.1. Optionally counter stain for 45 swith Gill's hematoxylin.

SECTION: Viral in situ hybridization
53.2. Wash with water for 5 min.

SECTION: Viral in situ hybridization
53.3. Mount the slides with fluoromount (Roth) by using a robotic coverslipper.

SECTION: Viral in situ hybridization
54.   
  

SECTION: ACE2 in situ hybridization
55. For ACE2 in situ hybridization proceed with the "RNAscope® Multiplex Fluorescent Reagent Kit v2 Assay":

  

The following changes were made using the USM-323100 protocol:

Page 14, step 5: a third xylene incubation step is implemented (5 min at RT).
Page 15, target retrieval: a digital heat plate is used instead of a steamer.
Page 17: a staining cuvette is used instead of the slide holder.

After finishing the steps for "FFPE sample preparation and pretreatment", continue with chapter 4 on page 28.

Page 31: Opal is used with a dilution of 1:1500.

Exemplary results are shown at step 41.