TITLE: HCR-FISH for Choanoflagellate Cultures
AUTHORS: Kayley Hake

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is a fluorescent </span><span style = "font-style:italic;">in situ </span><span>hybridization (FISH) technique for labeling 16S rRNA from specific bacteria in choanoflagellate cultures. It combines the fixation, cell mounting, and washing methods used in traditional FISH and catalyzed reporter deposition (CARD) FISH (</span><a href="https://aem.asm.org/content/65/8/3721.short" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Glöckner et al., 1999</span></a><span>; </span><a href="https://aem.asm.org/content/68/6/3094.short" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Pernthaler et al., 2002</span></a><span>), with the more permeablie </span><span style = "font-style:italic;">in situ </span><span>hybridization signal amplifcation technique of hybridization chain reaction (HCR) FISH (</span><a href="http://dev.biologists.org/content/143/19/3632" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Choi et al., 2016</span></a><span>; </span><a href="https://mbio.asm.org/content/7/5/e00796-16" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">DePas et al., 2016</span></a><span>). This is the first successful FISH method in choanoflagellates, and it provides a good foundation for future adaption for mRNA and other FISH methods in choanoflagellates. </span></div></div>

[STEPS]
?. [FIX Choanoflagellate culture]
?. [FILTER Choanoflagellate culture]
Using a 15ml glass microanalysis filter holder (Millipore Sigma), filter 250-1000µl of fixed culture onto a filter. To capture all choanoflagellate and free-living bacteria, use a 0.2µm pore size 25mm filter (GTTP02500, Millipore Sigma). To capture only choanoflagellate colonies, use a 5µm pore size 25mm filter (TMTP02500, Millipore Sigma). To help preserve morphology, apply the least amount of vacuum as possible, or let gravity filter the cultures.To assess density of the culture on the filter, carefully look at a filter under a cell culture microscope. Large choanoflagellate colonies are easy to visualize with 10-20x objectives, but smaller colonies are not. If it is hard to make out the choanoflagellates, stain the filters for 5 minutes in 0.1mg/mL Hoechst 33342 (Thermo Fischer) in sea water. Based on the density of the first filter you look at, adjust the amount of culture being added or keep the amount the same and finish filtering your fixed culture.Let filters air-dry face up (cells up) on clean whatman filter paper and label the rim with a pencil to mark the culture information that was filtered. Keep your labeling small, because you will need to add additional labels in the future.Filters can be stored at -20˚C until needed, or continued onto the next step. Filters are stable for many months at -20˚C and should be stored inside a clean piece of folded whatman filter paper in an air tight container.
?. [COAT Filters in agarose]
Prepare 0.1% low melt agarose with nuclease free H2O and sterile filter through a 0.2µm filter.  Allow the agarose to cool to 35-40˚C before using.Tape down a clean sheet of parafilm onto a desk.Pipette 2 ml of 0.1% low melt agarose onto the parafilm creating a puddle the size of a 25mm filter. Using tweezers to hold only the rim of the filter, dip the backside of the filter (side without cells) into the 0.1% low melt agarose first followed by the topside (side containing cells).Place the coated filter FACE DOWN (side containing cells) onto a clean portion of the taped down parafilm.Use separate puddles of 0.1% low melt agarose for different cultures to limit contamination between samples.Let filters dry at room temperature (1-2 hours). It is easy to tell when the 0.2µm filters are dry because the color will change from grey back to white. Soak the filters in 2ml of 96% ethanol for 1 min to float filters off of the parafilm. Carefully peel off the filters using tweezers to grab only the rim of the filter, and place the filter FACE UP on a clean piece of whatman filter paper to air-dry.Coated filters can be stored at 4˚C for a few months. Store filters inside a clean piece of folded whatman filter paper in an air tight container.
?. [HCR-FISH: PERMEABILIZATION]
Before you cut the filters for each sample or probe combination, label the rim of the 25mm filter in pencil on the side containing the cells with the sample number and approximate placement for each wedge. Write only the number, and provide more detail corresponding to those numbers in your lab notebook. Make sure to label in away that makes the filter orientation clear (ie. instead of 8 write 08).Using tweezers in one hand to hold the filter, carefully cut filters into the desired wedges using disecting scissors (typically 8-10 wedges per 25mm filter). Cut filters over a clean piece of whatman paper to collect wedges as they fall.Use tweezers to grab only the labeled rim of each wedge, place up to 10 wedges in a 2 ml tube with the point facing down (the flat bottom is important for proper distribution of filters in buffers). Prepare fresh CARD-FISH proteinase buffer in a separate tube (make 2 ml of Proteinase Buffer per 2 ml tube) and transfer into the tube containing the filters once it is made. Make sure all of the filters are submerged in the CARD-FISH Proteinase Buffer, and flick the bottom of the tube gently to remove any air bubbles on the filters.Incubate for 30 min. at 37˚C.Wash 2x for 1 min in nuclease free H2O at room temperature. (Be careful when pipetting to not touch the filters or accidently get them caught on the filter tip).Wash 1x in 98% EtOH and let filters air-dry on clean Whatman filter paper at room temperature.CARD-FISH Proteinse Buffer  AB1ReagentPer 2 ml20.5M EDTA0.2 ml31.0M Tris-Cl pH 8.00.2 ml4Nuclease free H2O1.6 ml5Lysozyme20 mg
AB1ReagentPer 2 ml20.5M EDTA0.2 ml31.0M Tris-Cl pH 8.00.2 ml4Nuclease free H2O1.6 ml5Lysozyme20 mg
?. [HCR-FISH: PROBE HYBRIDIZATION]
Transfer filters to a clean 2ml tube and add enough hybridiation buffer to completely cover the filters.Pre-hybridize filters in hybridization buffer for 30 min. at 45˚C. (Can keep filters grouped at this point)(Hybridization buffer recipe can be found in the "Before Start" section).Add 0.25µl of 100mM stock of HCR-FISH probes to 0.5ml of hybridization buffer to a new 2ml tube and vortex to mix. (Each probe combintion should be in it's own tube)(Information on how to design custom HCR-FISH probes can be found in our protocols.io called "16S rRNA probe design for HCR-FISH")Pre-warm hybridization buffer and probe solution to 45˚C.Add filters to their respective probe solutions.Incubate overnight at 45˚C.
?. [HCR-FISH: PROBE WASH]
Make fresh probe wash buffer with appropriate NaCl concentration based on the formamide% (See recipe below and the "Before Start" section for more information)( Add SDS last to prevent precipitation).Pre-warm fresh wash buffer to 48˚C. Replace hybridization and probe solution with 2ml of pre-warmed wash buffer to remove excess and unbound probe. Incubate for 1 hour at 48˚C.Remove probe wash buffer and wash filters 3 x 5 min. with 5X SSCT at room temperature.Wash Buffer (for 20% Formamide Concentration)  AB1ReagentPer 50 ml20.5M EDTA, pH 8.00.5 ml31.0M Tris HCl, pH 8.01.0 ml45M NaCl*2150 µl5Fill with nuclease free H2O to:50 ml620% SDS (w/v)**25 µl5x SSCT (For 40 ml)  AB1ReagentPer 40 ml220X SSC (nuclease free)10 ml310% Tween 20400 µl4Fill with nuclease free H2O to:40 ml
AB1ReagentPer 50 ml20.5M EDTA, pH 8.00.5 ml31.0M Tris HCl, pH 8.01.0 ml45M NaCl*2150 µl5Fill with nuclease free H2O to:50 ml620% SDS (w/v)**25 µl
AB1ReagentPer 40 ml220X SSC (nuclease free)10 ml310% Tween 20400 µl4Fill with nuclease free H2O to:40 ml
?. [HCR-FISH: FLUORESCENT HAIRPIN PREPARATION]
Filtersin 500µl of amplification buffer for 30 min. at room tempearture. (Amplification buffer recipe can be found in the "Before Start" sectionHairpin SolutionH1B)While the filters are in the amplification buffer, calculate how much fluorescent HCR hairpins you will need and prepare them for the next steps. HCR hairpins have the fluorophore attached to them and require two hairpins per color to self-assemble during the signal amplifcation step (See the diagram in the "Before Start"). HCR hairpins are pre-diluted in hairpin solution that can be purchased through Molecular Instruments or synthesized by hand following their protocols.In the King lab we have six different HCR hairpins that assemble in pairs into 3 different colors (Table below). For each hairpin color, their is an amplifier sequence that we've assigned. (For more information on amplifier sequences, see our protocol.io on how to design custom HCR-FISH probes called "16S rRNA probe design for HCR-FISH")HCR Hairpin Colors and Names In the signal amplification step we are preparing for, the hairpins will be diluted in amplification buffer. To calculate the amount of hairpins you need, you first need to calculate the amount of hairpin cocktail you will be making (amplification buffer + hairpin solution).To better explain, I will use an example experiement with 6 filters that have been hybridized with 3 probes requireing  3 colors (488, 594, and 647). For each filter, you will use 25µl of cocktail. So for 6 filters you will need 150µl of cocktaill. To limit the chance of running out in the end, add an additional 10µl to the cocktail making the final volume 160µl. You will use 1µl of hairpin solution for every 50µl of cocktial solution. This means for a cocktail with 160µl(160/50= 3.2) you need 3.2µl of each hairpin solution, and for cocktail 2, you need 4.2µl. Although you need to keep each hairpin solution separate, you can group the same hairpin solutions you need for both cocktails together resulting in the following total amount of hairpin solutions to be used in this experiement.Total amount of hairpin solutions needed: Once you calculate the amount of hairpins you need, pipette the amount of hairpin solution into individual tubes.To prepare the hairpins by snap cooling, heat the hairpins to 95˚C for 90 seconds and let them cool in the dark at room temperature for 30 min.While the hairpins are cooling, calculate the amount of amplification buffer you need by subtracting the volume of total hairpins solutions from the cocktail volume.While the filters are in the amplification buffer, calculate how much fluorescent HCR hairpins you will need and prepare them for the next steps. HCR hairpins have the fluorophore attached to them and require two hairpins per color to self-assemble during the signal amplifcation step (See the diagram in the "Before Start"). HCR hairpins are pre-diluted in hairpin solution that can be purchased through Molecular Instruments or synthesized by hand following their protocols.In the King lab we have six different HCR hairpins that assemble in pairs into 3 different colors (Table below). For each hairpin color, their is an amplifier sequence that we've assigned. (For more information on amplifier sequences, see our protocol.io on how to design custom HCR-FISH probes called "16S rRNA probe design for HCR-FISH")HCR Hairpin Colors and Names In the signal amplification step we are preparing for, the hairpins will be diluted in amplification buffer. To calculate the amount of hairpins you need, you first need to calculate the amount of hairpin cocktail you will be making (amplification buffer + hairpin solution).To better explain, I will use an example experiement with 6 filters that have been hybridized with 3 probes requireing  3 colors (488, 594, and 647). For each filter, you will use 25µl of cocktail. So for 6 filters you will need 150µl of cocktaill. To limit the chance of running out in the end, add an additional 10µl to the cocktail making the final volume 160µl. You will use 1µl of hairpin solution for every 50µl of cocktial solution. This means for a cocktail with 160µl(160/50= 3.2) you need 3.2µl of each hairpin solution, and for cocktail 2, you need 4.2µl. Although you need to keep each hairpin solution separate, you can group the same hairpin solutions you need for both cocktails together resulting in the following total amount of hairpin solutions to be used in this experiement.Total amount of hairpin solutions needed: Once you calculate the amount of hairpins you need, pipette the amount of hairpin solution into individual tubes.To prepare the hairpins by snap cooling, heat the hairpins to 95˚C for 90 seconds and let them cool in the dark at room temperature for 30 min.While the hairpins are cooling, calculate the amount of amplification buffer you need by subtracting the volume of total hairpins solutions from the cocktail volume.HCR Hairpin Colors and Names  ABCD1Hairpin FluorophoreAmplifier SequenceHairpin 1 SolutionHairpin 2 Solution2488B1B1H1B1H23594B2B2H1B2H24647B3B3H1B3H2In the signal amplification step we are preparing for, the hairpins will be diluted in amplification buffer. To calculate the amount of hairpins you need, you first need to calculate the amount of hairpin cocktail you will be making (amplification buffer + hairpin solution).To better explain, I will use an example experiement with 6 filters that have been hybridized with 3 probes requireing  3 colors (488, 594, and 647). For each filter, you will use 25µl of cocktail. So for 6 filters you will need 150µl of cocktaill. To limit the chance of running out in the end, add an additional 10µl to the cocktail making the final volume 160µl. You will use 1µl of hairpin solution for every 50µl of cocktial solution. This means for a cocktail with 160µl(160/50= 3.2) you need 3.2µl of each hairpin solution, and for cocktail 2, you need 4.2µl. Although you need to keep each hairpin solution separate, you can group the same hairpin solutions you need for both cocktails together resulting in the following total amount of hairpin solutions to be used in this experiement.Total amount of hairpin solutions needed: Once you calculate the amount of hairpins you need, pipette the amount of hairpin solution into individual tubes.To prepare the hairpins by snap cooling, heat the hairpins to 95˚C for 90 seconds and let them cool in the dark at room temperature for 30 min.While the hairpins are cooling, calculate the amount of amplification buffer you need by subtracting the volume of total hairpins solutions from the cocktail volume.Total amount of hairpin solutions needed:  ABC1FluorophoreHairpin SolutionAmount needed2488B1H13.2µl3488B1H23.2µl4594B2H13.2µl5594B2H23.2µl6647B3H13.2µl7647B3H23.2µlOnce you calculate the amount of hairpins you need, pipette the amount of hairpin solution into individual tubes.To prepare the hairpins by snap cooling, heat the hairpins to 95˚C for 90 seconds and let them cool in the dark at room temperature for 30 min.While the hairpins are cooling, calculate the amount of amplification buffer you need by subtracting the volume of total hairpins solutions from the cocktail volume.Calculations for the amount of Amplification Buffer needed  ABC1Cocktail VolumeMinus ( # of hairpins)x(amount of hairpin solution)Amount of Amplification Buffer2160 µl(6 hairpins)(3.2µl of hairpin solution)= 19.2µl140.8 µl
ABCD1Hairpin FluorophoreAmplifier SequenceHairpin 1 SolutionHairpin 2 Solution2488B1B1H1B1H23594B2B2H1B2H24647B3B3H1B3H2
ABC1FluorophoreHairpin SolutionAmount needed2488B1H13.2µl3488B1H23.2µl4594B2H13.2µl5594B2H23.2µl6647B3H13.2µl7647B3H23.2µl
ABC1Cocktail VolumeMinus ( # of hairpins)x(amount of hairpin solution)Amount of Amplification Buffer2160 µl(6 hairpins)(3.2µl of hairpin solution)= 19.2µl140.8 µl
?. [HCR-FISH: HAIRPIN HYBRIDIZATION]
After the hairpin solutions have cooled for 30 min., make the hairpin cocktail by adding all the respective hairpin solutions to the amount of amplification buffer. (ie. Add 3.2µl for each of the 6 hairpin solutions to 140.8 µl of amplification buffer.)In an air tight container, tape down a fresh piece of parafilm at the bottom.Drop out 25 µl of hairpin cocktail (amplification buffer + hairpin solution) onto the parafilm for each filter. Give enough space between the drops to fit the filters.Using tweezers to grab the labeled rim, carefully place each filter face down (cells down) on their respective drops.Add a wet kimwipe to the corner or side away from the filters in the chamber to keep it humid.Seal the container with parafilm and place it in the dark at room temperature.Incubate overnight.
?. [HCR-FISH: HAIRPIN WASH]
Using tweezers, carefully combine 6-10 filters into a 2 ml tube with 2 ml of Amplification Buffer.Incubate for 1 hour in the dark at room temperature to wash away unbound hairpins.Wash 2x 30 min. in 5x SSCT in the dark at room temperature.Wash 1x 10 min. in 5X SSCT + Hoechst 33342 diluted 1:1000 at room temperature in the dark.Wash 1 minute at room temperature in nuclease free H2O.Wash 1 minute in 96% EtOH.Let the filters dry on whatman paper.
?. [HCR-FISH: SAMPLE MOUNTING]
Using a pipette tip that has been cut at the end for viscous solutions, pipette a large smear of ProLong Diamond (Molecular Probes) onto a coverslip. For 24x40-1 coverslips (12-545-D Fisher), I place ~125µl of mounting solution. Using tweezers, carefully place filters cells down onto the smear of Prolong Diamond. (Mount 4-6 filters on a 24x40 coverslip)Place a similar smear of mounting solution onto a glass slide.Carefully flip the slide onto the coverslip by lowering the slide slowly from above until the mounting solutions from each side touch. Lower slowly to let the mounting solution spread out and prevent air bubbles.Leave slides to set overnight in the dark at room temperature.