May 11, 2026
RNAse free 4% Paraformaldehye in .1M PB preparation
Forked from 4% Paraformaldehye in .1M PB preparation
- 1HHMI Janelia Research Campus
Protocol Citation: Monique Copeland 2026. RNAse free 4% Paraformaldehye in .1M PB preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jj4kvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 11, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 316824
Keywords: perfusion solution, preparation, stream rna preservation, rnase, perfusion
Funders Acknowledgements:
HHMI
Abstract
Fixative for perfusion with down-stream RNA preservation and detection.
Materials
MATERIALS
ParaformaldehydeCatalog #19200
Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9932
Protocol materials
Sodium Hydroxide, 10.0NThomas ScientificCatalog #C871W32
ParaformaldehydeCatalog #19200
Corning™ Disposable Vacuum Filter/Storage Systems Fisher ScientificCatalog #09-761-5
Falcon Tube (50 mL)Fischer Scientific
Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9932
Sodium Phosphate Dibasic Dihydrate (USP/FCC/EP/BP), Fisher ChemicalFisher ScientificCatalog #S472-500
Paraformaldehyde prep
Add 400 mL of Nuclease-free water to a 1000 mL beaker
Add stir bar in beaker and set on hot plate at 50 °C
Add 5 drops (500ul) of 10N NaOH (from 60ml dropper)
Sodium Hydroxide, 10.0NThomas ScientificCatalog #C871W32
(this makes the solution basic to help dissolve the paraformaldehyde)
Weigh 20 g of Paraformaldehyde and add to MilliQ water.
(Stir about 01:00:00 or until all parafromaldehyde is dissolved and solution is clear)
ParaformaldehydeCatalog #19200
Make 1 Molarity (M) Phosphate buffer at PH 7.4
To make 100 mL 1M PB at PH 7.4 add 19 mL 1M Sodium phosphate Monobasic plus 81 mL 1M Sodium Dibasic dihydrate (store as stock at Room temperature )
Sodium Phosphate Dibasic Dihydrate (USP/FCC/EP/BP), Fisher ChemicalFisher ScientificCatalog #S472-500
Sodium Phosphate Dibasic Dihydrate (USP/FCC/EP/BP), Fisher ChemicalFisher ScientificCatalog #S472-500
Add 50 mL 1M PB PH 7.4 to paraformaldehyde solution.
Test PH and adjust solution to final desired 7.4pH using HCl or NaOH.
Top off to 500ml with MilliQ water.
Filter solution through .2um CA member filter.
Corning™ Disposable Vacuum Filter/Storage Systems Fisher ScientificCatalog #09-761-5
Label container with 'RNAse free 4% paraformaldehyed in .1M PB' and date. Store at 4 °C for up to 1 week.
(Can also be aliquoted into
Falcon Tube (50 mL)Fischer Scientific
and stored at -20 °C for 3 months.)