TITLE: 7: User-friendly protocol: Retina Tissue Sections RNA FISH
AUTHORS: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about SABER RNA FISH with retina tissue.</div></div>

[STEPS]
?. [Fixation]
Dissect neural retinas in PBS and fix in  (diluted from , Thermo Fisher #28908) for  at .
[ FA]
[ FA ampule]
0 Room temperature
?. [Preparing the slide]
Prepare PDL using  dissolved in , diluted from  with ddH2O.
[PDL (sigma P7886)]
[Borate buffer]
[stock (Thermo Scientific, #28341)]
?. [Preparing the slide]
Store PDL in aliquots at .
-20 °C
?. [Preparing the slide]
Apply PDL 8-well Ibidi chamber slide (Cat #80827) for at least .
?. [Preparing the slide]
Then remove PDL.
?. [Preparing the slide]
Dry the slide.
?. [Preparing the slide]
Wash once in ddH2O.
?. [Preparing the slide]
Dry again.
?. [Sectioning]
Place the PDL coated and fully dried slide inside the cryostat ahead of sectioning. Do not remove slide until all sections are in place or condensation will form that can impede adhesion.
?. [Sectioning]
Place sections inside wells using brushes, and flatten sections as much as possible to prevent folding when the slide is transferred to .
-20 Room temperature
?. [Sectioning]
After removing the slide from the chamber, spin at  for  (plate spinner centrifuge) 1-2 times to promote adhesion.
Centrifuge: 600 34
?. [Hybridization]
Wash sections 3× in PBSTw to remove OCT.
?. [Hybridization]
Wash sections  in PBSTw to remove OCT. (1/3)
?. [Hybridization]
Wash sections  in PBSTw to remove OCT. (2/3)
?. [Hybridization]
Wash sections  in PBSTw to remove OCT. (3/3)
?. [Hybridization]
Replace PBSTw with Whyb.
?. [Hybridization]
Place in hybridization oven set to , minimum .
43 °C
?. [In oven, set to 43°C]
Replace Whyb with Hyb1/Probe mixture pre-warmed to . Seal chamber with parafilm. Fill unused wells with Whyb to maintain humidity in the chamber sli.
43 °C
?. [In oven, set to 43°C]
Incubate  minimum.
?. [In oven, set to 43°C]
Replace Hyb1 with Whyb (quick wash to remove residual Hyb1).
?. [In oven, set to 43°C]
Wash 2× in Whyb.
?. [In oven, set to 43°C]
Wash  in Whyb. (1/2)
?. [In oven, set to 43°C]
Wash  in Whyb. (2/2)
?. [In oven, set to 43°C]
Wash 2× in .
[SSCT]
?. [In oven, set to 43°C]
Wash  in . (1/2)
[SSCT]
?. [In oven, set to 43°C]
Wash  in . (2/2)
[SSCT]
?. [In oven, set to 43°C]
Return to  (Pause point: sample can be stored in  or PBSTw at  for several weeks).
43 Room temperature
[SSCT]
4 °C
?. [Fluorescent detection]
Replace  with PBSTw (2 washes at ).
[SSCTw]
4 Room temperature
?. [Fluorescent detection]
Set oven to .
37 °C
?. [Fluorescent detection]
Transfer slide to  for hybridization and subsequent wash steps.
37 °C
?. [Fluorescent detection]
Once slide is warm, remove PBSTw and add Hyb2/fluor solution (prewarmed).
?. [Fluorescent detection]
Incubate at least  at .
37 °C
?. [Fluorescent detection]
Replace Hyb2 with PBSTw (prewarmed, quick wash).
?. [Fluorescent detection]
Wash 2× in PBSTw.
?. [Fluorescent detection]
Wash  in PBSTw. (1/2)
?. [Fluorescent detection]
Return to  (Pause point: Samples can be stored at  without obvious signal loss for at least 1 week).
37 Room temperature
4 °C
?. [Imaging]
Wash once in PBS and add mounting media until sections are covered (See below for notes on imaging).
?. [Serial detection (complete before repeating fluorescent detection)]
Remove glycerol mounting media with 3 or 4 PBSTw washes (~).
?. [Serial detection (complete before repeating fluorescent detection)]
Wash PBSTw. (1/4)
?. [Serial detection (complete before repeating fluorescent detection)]
Wash PBSTw. (2/4)
?. [Serial detection (complete before repeating fluorescent detection)]
Wash PBSTw. (3/4)
?. [Serial detection (complete before repeating fluorescent detection)]
Wash PBSTw. (4/4)
?. [Serial detection (complete before repeating fluorescent detection)]
Wash 3× in Displacement buffer at .
4 Room temperature
?. [Serial detection (complete before repeating fluorescent detection)]
Wash  in Displacement buffer at . (1/3)
4 Room temperature
?. [Serial detection (complete before repeating fluorescent detection)]
Wash  in Displacement buffer at . (2/3)
4 Room temperature
?. [Serial detection (complete before repeating fluorescent detection)]
Wash  in Displacement buffer at . (3/3)
4 Room temperature
?. [Serial detection (complete before repeating fluorescent detection)]
Wash 3× in PBSTw.
?. [Serial detection (complete before repeating fluorescent detection)]
Wash  in PBSTw. (1/3)
?. [Serial detection (complete before repeating fluorescent detection)]
Wash  in PBSTw. (2/3)
?. [Serial detection (complete before repeating fluorescent detection)]
Wash  in PBSTw. (3/3)
?. [Fixation]
Embed retinas in a 1:1 (v/v) mixture of  and OCT, frozen in ethanol bath (200 proof ethanol cooled in dry ice). Be careful to ensure that ethanol bath does not contact the embedded retina or the embedding solution.`
[sucrose (in 1×PBS)]
?. [Fixation]
Store frozen blocks at  prior to sectioning.
-80 °C