Paraformaldehyde and Glyoxal Fixation Protocol

Paraformaldehyde (Merck, #8.18715) was dissolved in PBS (pH 7.4) at a final concentration of 4%. The glyoxal fixative solution was prepared according to published protocol (Richter et al., 2018 (link)). Briefly, 28 ml of ddH₂O, 7.89 ml absolute of ethanol (analysis grade), 3.13 ml of glyoxal (Sigma-Aldrich, #128465), and 0.3 ml of acetic acid (Sigma-Aldrich, #A6283) were mixed well by vortex. After adjusting to pH 4.0 with 1 N of NaOH, the solution was filled up to 40 ml with ddH₂O. The final concentration of glyoxal was 3%. Both fixative solutions were prepared on the day of the experiment and kept cool until use.