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[BEFORE_START]
Prepare wash buffer at same time as setting up hybridization.

[GUIDELINES]
<em><strong>Sample Collection</strong></em><br /><br /><em>Biosphere 2 Samples</em><br /><br />Materials<br />• Acid-Washed Glass Bottles (Enough for samples + 1 for collecting ocean water)<br />• 16% Formaldehyde<br />• 200 Proof Ethanol <br />• Ice &amp; Cooler for transport (or 4°C equivalent)<br />• Serological Pipettes (25mL)<br />• Electronic Pipettor <br /><br />Protocol<br />1. Submerge inverted capped acid-washed glass below the surface of the B2 ocean.<br />2. Remove cap while submerged and allow water to fill glass bottle. Swirl and empty.<br />3. Repeat Steps 1 &amp; 2, but this time keep the seawater. This is your sample<br />4. Call B2 Energy Center to obtain B2 ocean temperature &amp; salinity at time of sampling.<br />5. Add 70mL of 16% Formaldehyde to a new acid-washed glass bottle.<br />6. Add your sample to the bottle to q. to 1 liter.<br />7. Again repeat Steps 1 &amp; 2 and keep the seawater.<br />8. Add 500mL of 200 Proof Ethanol to a new acid-washed glass bottle.<br />9. Add your sample to the bottle to q. to 1 liter.<br />10. Right before you leave the B2 Ocean, repeat Steps 1 &amp; 2 and keep the seawater.<br />11. Add 1 liter of seawater to a new acid-washed glass.<br /><br /><em><strong>Filtration Process</strong></em><br /><br /> Materials<br />• 3-place filtration manifold (stainless steel or polyurethane)<br />• 3 glass microanalysis filter holder assemblies 47mm (tower, clamp, and base)<br />• 1 glass microanalysis filter holder assembly 90mm (tower, clamp, and base)<br />• 90mm GF/D Filter<br />• 47mm cellulose nitrate 0.45µm supor filter<br />• 47mm 0.2µm GTTP Isopore membrane filters<br />• 2 2 liter glass flasks with arm<br />• Tubing<br />• Vacuum pump<br /><br />Protocol<br />1. Attach a 2 liter glass flask with arm to the vacuum pump using tubing. <br />2. Set up the 90mm filter holder assembly and add the 90mm GF/D filter. <br />3. Add your sample to the tower and turn on the vacuum to begin filtration. Make sure the vacuum pressure does not exceed 5 in Hg. The filtered product is called the pre-filtered product.<br />4. Set up the 47mm filter holder assembly with the flask and add the 0.2um GTTP Isopore membrane filters shiny-side up.<br />5. Filter the 15mL pre-filtered product per membrane filter (x3). Filter 100mL pre-filtered product per membrane filter (x9).<br />6. Use tweezers to remove membrane filters from the filter holder assembly and put on blotting paper in the dark shiny side up.<br />7. Once the membrane filter is dry, store in a petri dish.<br /><br /><em><strong>CARD FISH</strong></em><br /><em>Reconstituting FISH probes</em><br /><br /> Materials<br />• Lyophilized Probe<br />• PCR water<br />• Ice &amp; Ice Bucket<br />• Nanodrop System<br /><br />Protocol<br />1. Add 100µL PCR water to lyophilized probe.<br />2. Incubate on ice for &gt; 2 hours.<br />3. Finger-flick a few times and shake down material to bottom of tube. (NOTE: you do not want to vortex because HRP enzyme attached to the probe may detach)<br />4. Use 1.5µL of reconstituted probe and check the UV at 260 and 404 wavelengths on the Nanodrop. <br />5. Add PCR water depending on Nanodrop results.<br /><br /><em>Embedding Samples</em><br />***NOTE: For microscopy samples only!<br /><br /> Materials<br />• Low Gelling Point Agarose<br />• Parafilm<br />• Glass Slide<br />• 80-96% Ethanol<br />• Tweezers (Non-fine)<br />• 2 Large-sized Petri Dishes<br />• Sample filtered on 47mm 0.2µm GTTP Isopore membrane filters<br /><br />Protocol<br />1. Pre-warm a petri-dish. <br />2. Boil 0.1% low gelling point agarose and pour into the pre-warmed petri-dish.<br />3. Let agarose cool down to 35-40°C.<br />4. Cover glass slides with layer of parafilm so that there is an even surface.<br />5. 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.<br />6. Let dry at room temperature.<br />7. Remove filter from slide surface by soaking parafilm covered slide in petri-dish filled with 80-96% Ethanol.<br />8. Air-dry filter on a kimwipe.<br /><br /><em>Pre-Treatment</em><br /><br /> Materials<br />• Lysozyme Solution (10mg/mL)<br />• Recipe (10mL):<br />    • 1mL 0.5M EDTA pH 8.0<br />    • 3mL 1M Tris-HCl pH 8.0<br />    • 6mL MQ water (autoclaved)<br />    • 100mg Lysozyme (Fisher Cat#BP535-5)<br />• 0.01M HCl<br />• Water Bath<br />• Tweezers (Non-fine)<br />• Sterile Scalpel<br />• Blotting paper<br />• Pencil<br />• 2 Medium-sized Petri Dishes<br />• Sample filtered on 47mm 0.2µm GTTP Isopore membrane filters<br /><br />Protocol<br />1. Turn on water bath and pre-warm to 37°C and sterilize tweezers with 70% EtOH.<br />2. 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.<br />3. Make fresh lysozyme solution and pour it into a petri dish. <br />4. To <em>permeabilize</em> your sample, submerge your samples in the lysozyme solution. Wrap parafilm around the edge of the petri-dish.<br />5. Put the petri-dish into the 37°C water bath for 1 hour.<br />6. While waiting for your incubation, pour 0.01M HCl in a new petri-dish.<br />7. Once your incubation is over, put your sample filters in a strainer and rinse off any residual lysozyme solution with distilled water.<br />8. To <em>inactivate of endogenous peroxidases</em>, submerge your sample in the 0.01M HCl and incubate for 15 minutes.<br />9. Again, put your sample filters in a strainer and rinse off any residual lysozyme solution with distilled water.<br />10. Air dry on a kimwipe.<br /><br /><em>Hybridization</em><br /><br /> Materials<br />• Probe<br />• Hybridization Chamber Mix<br />    • Recipe (2mL)<br />        • % Formamide <br />        • q. to 2mL with MQ water<br />• Hybridization Buffer<br />    • Recipe (refer to Elke’s recipe)<br />• Hybridization Ovens<br />• Tweezers (Non-fine)<br />• Sterile Scalpel<br />• Blotting paper<br />• Pencil<br />• Sample filtered on 47mm 0.2µm GTTP Isopore membrane filters<br />• 50mL falcon tubes<br />• Styrofoam 50mL falcon tube holder<br />• Cardboard<br />• Glass slides<br />• Kimwipes<br />• Parafilm<br /><br /><img src="https://s3.amazonaws.com/pr-journal/kdc2xe.fish" alt="" width="239" height="500" /><br /><br />Protocol<br />1. Pre-heat hybridization ovens to 46°C and 48°C.<br />2. Create a hybridization humidity chamber setup (refer to <em>Fig.</em> <em>1</em>).<br />3. Based on the probe you plan to use for hybridization, determine the % formamide to use. <br />4. Create the individual hybridization humidity chambers (<em>Fig. 2</em>) by inserting a kimwipe soaked in the correct <strong>2mL % formamide-water mix (Hybridization Chamber Mix)</strong>. Cover glass slides evenly with parafilm. Do NOT add slide to individual chamber yet.<br />a. eg. If probe needs 35% formamide, create hybridization chamber mix that is 35% formamide (700µL formamide, 1300µL MQ water)<br />5. 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 “•”. <br />a. NOTE: The “•” indicates the orientation of the filter, once the “shine” is no longer obvious<br />6. Mix <strong>hybridization buffer</strong> with <strong>probe working solution</strong> [50ng DNA µl<sup>-1</sup>] in a 300:1 ratio.<br />7. Dip each filter completely into the <strong>Hybridization mix</strong> and place filters face-up on the parafilm covered glass slide. Spread the rest of the solution evenly onto the filters.<br />8. Put glass slide horizontally into individual hybridization humidity chamber with corresponding % formamide to probe.<br />9. Incubate at 46°C overnight.<br /><br /><em>Washing</em><br />*** Prepare wash buffer at same time as setting up hybridization<br /><br /> Materials<br />• Washing Buffer <br />    • Recipe (refer to Elke’s recipe)<br />• 1x PBS<br />• H<sub>2</sub>O<sub>2</sub><br />• Amplification Buffer<br />    • Recipe (refer to Elke’s recipe)<br />• Alexa 488<br />• EtOH<br />• Ice<br />• Tweezers (Non-fine)<br />• Large-sized Petri Dishes<br />• 50mL Falcon Tubes <br />• Glass slides<br />• Kimwipes<br />• Parafilm<br /><br />Protocol<br /><em>Prior Day</em><br />1. Make 50mL Washing Buffer in 50mL falcon tube with corresponding % formamide to probe.<br />2. Warm at 48°C overnight.<br /><em>Day Of</em><br />3. Cover glass slides evenly with parafilm. Set aside until later use.<br />4. Remove filters from individual humidity chambers and put in corresponding % formamide pre-warmed <strong>Washing Buffer</strong>. Incubate at 48°C for 10 minutes.<br />5. Pour 1x PBS in a Large-sized petri dish.<br />6. Transfer filters to 1x PBS and incubate for 15 minutes at room temperature.<br />7. Get <strong>Ice</strong> and put H<sub>2</sub>O<sub>2</sub>, Amplification Buffer, Alexa 488 in covered ice bucket.<br />8. Mix 1µL H<sub>2</sub>O<sub>2</sub> with 199µL 1x PBS.<br />9. Create the <strong>Substrate mix</strong> in a 1000 <strong>Amplification Buffer</strong>: 10 <strong>diluted H<sub>2</sub>O<sub>2</sub></strong> : 3.3 <strong>Alexa 488</strong> ratio. Keep in covered ice bucket until ready to use. <br />a. Eg. Substrate Mix: 1000µL Amplification Buffer, 10µL diluted H2O2, 3.3µL Alexa 488.<br />10. Take filters out of 1x PBS and quickly wipe off excess 1x PBS on kimwipe. Make sure NOT to touch to cell-side to the kimwipe.<br />11. Dip filters into the <strong>Substrate mix</strong> and put on parafilm covered slide. Spread the rest of the mix evenly onto the filters.<br />12. Put slides into large-sized petri dishes and seal petri dish with parafilm and put in 46°C for 45 minutes in the dark.<br />13. Dry filters on kimwipe and put in 1x PBS for 10 minutes at room temperature in the dark.<br />14. Pour MQ water into a Large-sized petri dish and pour 96% EtOH into a different Large-sized petri dish.<br />15. Transfer filters to MQ water and cover in dark for 1 min at room temperature.<br />16. Transfer filters to 96% EtOH and cover in dark for 1 min at room temperature.<br />17. Put on kimwipe on a paper towel and let dry covered in the dark.<br />18. Immediately begin resuspension step! NOTE: If for microscopy, you can store filters at -20°C.<br /><br /><em><strong>Resuspension of Cells from Filters</strong></em><br /><br /> Materials<br />• 150mM NaCl<br />• 10% Tween 80 Ampules<br />• 0.2µm syringe filter<br />• Luer-lok syringe<br />• Large-sized petri dish<br />• 2mL centrifuge tubes<br />• Tape <br />• Incubator<br />• Vortexers<br /><br />Protocol<br />1. Pre-heat incubator to 37°C.<br />2. 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.<br />3. 0.2µm filter sterilize the 150mM NaCl. <br />4. Make <strong>Resuspension Buffer</strong> (30mL 150mM NaCl; 160µL 10% Tween 80)<br />5. Put 1.5mL Resuspension Buffer per 2mL centrifuge tube and put filter in centrifuge tube.<br />6. Incubate centrifuge tube for 30 minutes at 37°C shaking horizontally.<br />7. Tape Tubes to vortexer horizontally (6 tubes per for vortexer). Shake in dark for 15 minutes at 2500 rpm.<br />8. Remove filter (BUT keep filter just in case). The cells should now be in the buffer!!<br /><br />.<br /><br /><br /><br /><br />

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