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HCR DNA-FISH
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Prepare necessary buffers as described in :doc:`/recipes/fish/fish_buffers`
Expected time: 2 days

Fixation
========
1. Steps 1-5 of :doc:`antibody_staining`

Hybridization
=============
1. Preheat Hybridization Buffer (HYB) to 37 degrees Celsius
2. Incubate cells at room temperature for 30 minutes in 500 uL of 20% glycerol in PBS
3. Flash freeze the cells

   a. Place coverslips in liquid nitrogen for 30 seconds
   b. Remove and let thaw for 1 minute

.. warning::
   Liquid Nitrogen can shatter the coverslip; be careful when freezing

4. Incubate cells at RT for 20 mins in 500 uL of 20% glycerol in PBS
5. Repeat Step 4
6. Incubate cells at RT for 5 mins in 500 uL of .1 N HCl
7. Rinse cells at RT with 500 uL of 2X SSC

   a. Repeat x2

8. Incubate cells at 37 degrees C in 500 uL of HYB for 30 mins
9.  Remove HYB and add 100 uL of probe+HYB mixture
10.  Incubate at 78 degrees C for 5 mins
11.  Incubate **overnight** at 37 degrees C in a humid environment

Probe Preperation
=================
1. Preheat Hybridization Buffer (HYB) to 37 degrees Celsius
2. Dilute probe stock with HYB to 10 pmol per 100 uL per reaction
3. Heat probe+HYB mixture at 70 degrees C for 5 mins
4. Chill on ice until use

Post-Hybridization
==================
1. Preheat Hybridization Buffer (HYB) to 37 degrees Celsius
2. Preheat Probe Wash Buffer to 37 degrees C
3. Remove probe+HYB, then wash with 500 uL of Probe Wash Buffer

   a. Repeat

4. Wash at RT with 500 uL of 5X SSCT for 5 mins

   a. Repeat

5. Wash at RT with 500 uL of PBS for 5 mins
6. Proceed to imaging