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 ) .
