FISH Protocol for FISH & FLOW
Submerge inverted capped acid-washed glass below the surface of the B2 ocean.
Remove cap while submerged and allow water to fill glass bottle.
Swirl and empty.
Repeat Steps 1-3, but this time keep the seawater.
Call B2 Energy Center to obtain B2 ocean temperature & salinity at time of sampling.
Add 70mL of 16% Formaldehyde to a new acid-washed glass bottle.
Add your sample to the bottle to q. to 1 liter.
Again repeat Steps 1-3 and keep the seawater.
Add 500mL of 200 Proof Ethanol to a new acid-washed glass bottle.
Add your sample to the bottle to q. to 1 liter.
Right before you leave the B2 Ocean, repeat Steps 1-3 and keep the seawater.
Add 1 liter of seawater to a new acid-washed glass.
Attach a 2 liter glass flask with arm to the vacuum pump using tubing.
Set up the 90mm filter holder assembly and add the 90mm GF/D filter.
Add your sample to the tower and turn on the vacuum to begin filtration.
Set up the 47mm filter holder assembly with the flask and add the 0.2um GTTP Isopore membrane filters shiny-side up.
Filter the 15mL pre-filtered product per membrane filter (x3).
Filter 100mL pre-filtered product per membrane filter (x9).
Use tweezers to remove membrane filters from the filter holder assembly and put on blotting paper in the dark shiny side up.
Once the membrane filter is dry, store in a petri dish.
Add 100µL PCR water to lyophilized probe.
Incubate on ice for > 2 hours.
Finger-flick a few times and shake down material to bottom of tube.
Use 1.5µL of reconstituted probe and check the UV at 260 and 404 wavelengths on the Nanodrop.
Add PCR water depending on Nanodrop results.
Pre-warm a petri-dish.
Boil 0.1% low gelling point agarose and pour into the pre-warmed petri-dish.
Let agarose cool down to 35-40°C.
Cover glass slides with layer of parafilm so that there is an even surface.
Using sterile tweezers, dip filter with both sides in the agarose and place it face-down (ie. shiny-side/bacteria-side down!) onto the parafilm-covered slide.
Let dry at room temperature.
Remove filter from slide surface by soaking parafilm covered slide in petri-dish filled with 80-96% Ethanol.
Air-dry filter on a kimwipe.
Turn on water bath and pre-warm to 37°C and sterilize tweezers with 70% EtOH.
While the water bath is warming up, take out a piece of blotting paper.
Put your sample filter shiny-side up on the blotting paper.
Cut your sample filters in half using sterile scalpel and sterile tweezers to stabilize.
Mark the filter piece using a pencil.
Make fresh lysozyme solution and pour it into a petri dish.
To permeabilize your sample, submerge your samples in the lysozyme solution.
Wrap parafilm around the edge of the petri-dish.
Put the petri-dish into the 37°C water bath for 1 hour.
While waiting for your incubation, pour 0.01M HCl in a new petri-dish.
Once your incubation is over, put your sample filters in a strainer and rinse off any residual lysozyme solution with distilled water.
To inactivate of endogenous peroxidases, submerge your sample in the 0.01M HCl and incubate for 15 minutes.
Again, put your sample filters in a strainer and rinse off any residual lysozyme solution with distilled water.
Air dry on a kimwipe.
Pre-heat hybridization ovens to 46°C and 48°C.
Create a hybridization humidity chamber setup.
Based on the probe you plan to use for hybridization, determine the % formamide to use.
Create the individual hybridization humidity chambers (Fig. 2 in guidelines) by inserting a kimwipe soaked in the correct 2mL % formamide-water mix (Hybridization Chamber Mix).
Cover glass slides evenly with parafilm.
Do NOT add slide to individual chamber yet.
Take out a piece of blotting paper.
Put your sample filter shiny-side up on the blotting paper.
Cut your sample already halved filters into eighths using sterile scalpel and sterile tweezers to stabilize.
Number the filter pieces using a pencil with a “#” followed by “•”.
Mix hybridization buffer with probe working solution [50ng DNA µl-1] in a 300:1 ratio.
Dip each filter completely into the Hybridization mix and place filters face-up on the parafilm covered glass slide.
Spread the rest of the solution evenly onto the filters.
Put glass slide horizontally into individual hybridization humidity chamber with corresponding % formamide to probe.
Incubate at 46°C overnight.
Make 50mL Washing Buffer in 50mL falcon tube with corresponding % formamide to probe.
Warm at 48°C overnight.
Cover glass slides evenly with parafilm.
Set aside until later use.
Remove filters from individual humidity chambers and put in corresponding % formamide pre-warmed Washing Buffer.
Incubate at 48°C for 10 minutes.
Pour 1x PBS in a Large-sized petri dish.
Transfer filters to 1x PBS and incubate for 15 minutes at room temperature.
Get Ice and put H2O2, Amplification Buffer, Alexa 488 in covered ice bucket.
Mix 1µL H2O2 with 199µL 1x PBS.
Create the Substrate mix in a 1000 Amplification Buffer: 10 diluted H2O2 : 3.3 Alexa 488 ratio.
Take filters out of 1x PBS and quickly wipe off excess 1x PBS on kimwipe.
Dip filters into the Substrate mix and put on parafilm covered slide.
Spread the rest of the mix evenly onto the filters.
Put slides into large-sized petri dishes and seal petri dish with parafilm and put in 46°C for 45 minutes in the dark.
Dry filters on kimwipe and put in 1x PBS for 10 minutes at room temperature in the dark.
Pour MQ water into a Large-sized petri dish and pour 96% EtOH into a different Large-sized petri dish.
Transfer filters to MQ water and cover in dark for 1 min at room temperature.
Transfer filters to 96% EtOH and cover in dark for 1 min at room temperature.
Put on kimwipe on a paper towel and let dry covered in the dark.
Immediately begin resuspension step!
Pre-heat incubator to 37°C.
Pour 1x PBS in a Large-sized petri dish.
Transfer hybridized filters to 1x PBS and incubate for 15 minutes in the dark at room temperature.
0.2µm filter sterilize the 150mM NaCl.
Make Resuspension Buffer (30mL 150mM NaCl; 160µL 10% Tween 80).
Put 1.5mL Resuspension Buffer per 2mL centrifuge tube and put filter in centrifuge tube.
Incubate centrifuge tube for 30 minutes at 37°C shaking horizontally.
Tape Tubes to vortexer horizontally (6 tubes per for vortexer).
Shake in dark for 15 minutes at 2500 rpm.
Remove filter (BUT keep filter just in case).
