Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Formaldehyde fixation

HG Helena García-Castro
NK Nathan J. Kenny
MI Marta Iglesias
PÁ Patricia Álvarez-Campos
VM Vincent Mason
AE Anamaria Elek
AS Anna Schönauer
VS Victoria A. Sleight
JN Jakke Neiro
AA Aziz Aboobaker
JP Jon Permanyer
MI Manuel Irimia
AS Arnau Sebé-Pedrós
JS Jordi Solana
request Request a Protocol
ask Ask a question
Favorite

For each fixation, we took 100–200 μL of previously trypsinized cells and resuspended them in 8 mL of buffer (1× PBS 1% BSA) in a 15-mL Falcon tube. We added 2 mL of previously prepared formaldehyde (FA) stock diluted in 1× PBS (at 0.5%, 2.5%, 5%, 10%, or 20% FA in PBS), to result in a final concentration of 0.1%, 0.5%, 1%, 2%, or 4% in 10 mL total volume. The Falcon tubes were immediately gently shaken to homogenize the FA concentration. Samples were then incubated for 10 min at 4 °C in a see-saw shaker at 35–45 rpm. We then centrifuged twice at 1000g for 5 min (4 °C) to remove FA. After each centrifugation step, the supernatant was discarded. Fixed cells were resuspended in 5–7 mL of buffer (1× PBS 1% BSA), after the first centrifugation, and in 1 mL of TRIzol after the second, for RNA extraction as described below.

Copyright and License information: The Author(s) ©2021
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A