TITLE: FISH in Ashbya
AUTHORS: sierrajc

[DESCRIPTION]
Abstract: This is Fluorescent in situ Hybridization for looking at transcripts in  Ashbya. If using this to look at RNA, then try to use all RNase-free material at the RNA bench. This was adapted from Therese Gerbich's protocol.

Keywords: Ashbya, Fluorescence, RNA,  DNA

[STEPS]
SECTION: Day 1
1. Grow 100mL culture of Ashbya from dirty spores, seeded at2-3 µL spores/mL culture at 30 deg for 15 hours in 1000 ml baffled flask. Start around 4.30 PM.
SECTION: Day 2
2. Add 10ml of 37% formaldehyde (final 3.7%) and shake for 1hr at 30 deg. Alternatively, first, spin cells down in 15 mL tubes, resuspend in 9 mL used media and add 1mL formaldehyde, to decrease PFA waste.
SECTION: Day 2
3. Spin in 15 ml conicals (Important! Do NOT use 50 mL tubes) at 400 rpm in tabletop centrifuge for 5 mins, discard supernatant in formaldehyde waste. Aspirate using 5 mL pipette vertically to avoid disturbing pellet.
SECTION: Day 2
4. Wash 2x with 5 mL ice-cold Buffer B. Make sure you have a visible white pellet at the end of this step. During this time make fresh spheroplasting buffer
SECTION: Day 2
5. Resuspend in 1 ml spheroplasting buffer and transfer to a new 1.5 ml microcentrifuge tube. 
SECTION: Day 2
6. Add 100 𝞵l zymolyase (15 mg/ml) and incubate on a nutattor at 37 deg. Check digestion every 5- 10 mins until cells phase dark. (This varies by day and by strain. Do not over-digest or cells will lyse)



 


SECTION: Day 2
7. Once phase dark, spin at 2000 rpm (digested cells are very fragile) for 2 mins in benchtop microfuge, wash 2x with 1 mL ice-cold buffer B. At this stage, the cells will become hard to pellet. Aspirate around suspended clumps. Setting the tube in a stand for 2 minutes before aspirating media helps decrease cell loss. Don’t worry about saving the hard-to-pellet cells, they are usually fragments of cells. 
SECTION: Day 2
8. If doing proteinase k digestion
SECTION: Day 2
8.1. Add a solution of 2.5 𝞵l Proteinase K in 500 𝞵l wash buffer to pellet and tap to mix.
SECTION: Day 2
8.2. Let cells sit in the proteinase k solution for 5 minutes at room temperature. Don’t let reaction proceed beyond this or cells will lyse.
SECTION: Day 2
8.3. Spin at 2000 rpm for 2 mins and remove supernatant.
SECTION: Day 2
8.4. Add 500 𝞵l of 1mM PMSF in wash buffer to the cells, invert, and let it sit for 5 minutes. This needs to be made fresh every time as PMSF is very unstable in aqueous solutions. Check that PMSF hasn’t crashed out of solution in the stock.
SECTION: Day 2
8.5. Wash once with wash buffer. Add 1 ml 70% RNAse free EtOH leave overnight at 4 deg.  Can be left in EtOH for a week. 
SECTION: Day 2
9. Spin final time to remove Buffer B and resuspend in 1 ml 70% RNAse free EtOH leave overnight at 4 deg. Cells should be in RNAse free ethanol at 4 deg for at least 4 h. The cells may be stored in EtOH at 4 deg for up to a week.
SECTION: Day 3 or Evening of Day 2
10. Prepare hybridization solution: 100 𝞵l hybridization buffer with 1-3 𝞵l probe. Vortex to mix and centrifuge to remove any droplets. 
SECTION: Day 3 or Evening of Day 2
10.1. Warm the hybridization solution to room temperature before opening
SECTION: Day 3 or Evening of Day 2
10.2. For initial tests of probes, it is best to start 4 separate hybridization reactions by adding 1 𝞵l each of the 1:10, 1:20, 1:50, 1:100 working dilutions to see which is optimal. CLN3 and BNI1 seem to work well using 1-2 𝞵lof 25 𝞵M probe
SECTION: Day 3 or Evening of Day 2
11. Centrifuge cells at 2000 rpm for 2 mins and remove ethanol. 
SECTION: Day 3 or Evening of Day 2
12. Resuspend in 1 ml of wash buffer let stand at room temperature 2-5 mins
SECTION: Day 3 or Evening of Day 2
13. Centrifuge samples and remove wash buffer. Add hybridization solution and incubate in the dark overnight at 37 deg on a nutattor.
SECTION: Day 4 or Day 3
14. Add 1 ml wash buffer to the sample, invert to mix, centrifuge (2000 rpm for 2 mins), and remove supernatant. 
SECTION: Day 4 or Day 3
15. Add 1 mL wash buffer and incubate at 37 deg for 30 min (to save time can add Hoechst to wash buffer)
SECTION: Day 4 or Day 3
16. If not performing GFP booster
SECTION: Day 4 or Day 3
16.1. Spin down cells, remove supernatant, resuspend in 500 𝞵l wash buffer (can be skipped). 
SECTION: Day 4 or Day 3
16.2. Add 1𝞵l Hoescht and incubate in the dark at room temperature for 15 min. (unless done previously  in step 15)
SECTION: Day 4 or Day 3
16.3. Wash with 500 𝞵l wash buffer. Remove as much supernatant as possible. At this point, you may not even see a pellet. That’s okay, as long as you had a small pellet till the previous step, you’ll see cells later.
SECTION: Day 4 or Day 3
16.4. Add enough mounting medium to roughly equal the volume of cells. Use a minimum of 20 𝞵l. Can increase if there are a lot of cells. Scrape the walls to get out the last of the cells, but make sure you don’t make bubbles. Pipette up and down slowly to mix.
SECTION: Day 4 or Day 3
16.5. Mount 20 𝞵l on coverslip then place the slide on top. For coverslip use 22x50mm. (This is important to not lose cells). Gently and flatten with a notebook with something heavy on the slide to help flatten Ashbya.
SECTION: Day 4 or Day 3
16.6. After about 10-15 mins of flattening, seal with nail polish. Slides may be stored at -20 deg.
SECTION: Day 4 or Day 3
17. If performing GFP booster
SECTION: Day 4 or Day 3
17.1. Wash 2x with 250ul 1x-PBS + 1 mg/ml BSA. Resuspend a final time in 250 𝞵l PBS + BSA incubate in the dark at room temperature for 30 min to block.
SECTION: Day 4 or Day 3
17.2. Wash 2x with 250 𝞵l PBS+BSA
SECTION: Day 4 or Day 3
17.3. Resuspend in 200 𝞵l PBS +BSA and add 1 l𝞵 GFP booster and incubate overnight in the dark at 4 deg 
SECTION: Day 4 or Day 3
17.4. On the next day, wash 2X with 250 𝞵l PBS+BSA
SECTION: Day 4 or Day 3
17.5. Resuspend in 500 𝞵l PBS+BSA and 1ul Hoechst and incubate in the dark at room temperature for 30 min. 
SECTION: Day 4 or Day 3
17.6. Wash with 500ul PBS + BSA, remove as much supernatant as possible. 
SECTION: Day 4 or Day 3
17.7. Add enough mounting media to roughly equal the volume of the cells. Use a minimum of 20 𝞵l.
SECTION: Day 4 or Day 3
17.8. Mount 20 𝞵l on coverslip then place the slide on top. For coverslip use 22x50mm. (This is important to not lose cells). Gently and flatten with a notebook with something heavy on the slide to help flatten Ashbya.
SECTION: Day 4 or Day 3
17.9. After about 10-15 mins of flattening, seal with nail polish. Slides may be stored at -20 deg.