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PFA is superior to glyoxal in preserving oocyte, embryo, and stem cell proteins evidenced by super-resolution microscopical surveys of epitopes

Abstract

Purpose

Chemical fixation is a critical step to retaining cellular targets as naturally as possible. Recent developments in microscopy allow sophisticated detection and measuring techniques with which spatio-temporal molecular alterations are conceivable. In this study, we compare two members of aldehyde fixatives [i.e., glyoxal (Gly) and paraformaldehyde (PFA)] to determine whether Gly, a less toxic dialdehyde fixative that is considered to retain immunoreactivity could provide a successful and consistent cell fixation in favor of PFA in various cell preparations and types.

Methods

We document the fixation competence of Gly and PFA side-by-side (with or without Triton X-100 permeabilization) in live- and fixed-cell preparations in mouse oocytes, embryos, and human somatic cells (human umbilical cord-derived mesenchymal stromal cells) using protein quantification by Western blot assay and super-resolution microscopy.

Results

Although Gly seemed to act faster than PFA, catastrophic consequences were found not acceptable, especially in oocytes and embryos. Due to cell lysate and immunocytochemistry surveys, it was obvious that PFA is superior to Gly in retaining cellular proteins in situ with little/no background staining. In many samples, PFA revealed more reliable and consistent results regarding the protein quantity and cellular localization corresponding to previously defined patterns in the literature.

Conclusion

Although the use of Gly is beneficial as indicated by previous reports, we concluded that it does not meet the requirement for proper fixation, at least for the tested cell types and proteins. However, PFA alone with no addition of TX displayed a significant cytoplasmic loss by generating membrane blebs during fixation.

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Abbreviations

3D:

Three-dimensional

ACRPB:

Acrosin-binding protein

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

CTCF:

Corrected total cell fluorescence

ddH2O:

Double distilled water

DIC:

Differential interference contrast

DMEM:

Dulbecco’s modified Eagle’s medium

F-actin:

Filamentous actin

FA:

Formaldehyde

FITC:

Fluorescein isothiocyanate

Gly:

Glyoxal

GV:

Germinal vesicle

hCG:

Human chorionic gonadotropin

hUC-MSC:

Human umbilical cord-derived mesenchymal stem cell

ICC:

Immunocytochemistry

IHC:

Immunohistochemical

mEmbryos:

Mouse embryos

MI:

Meiosis-I

mOocyte:

Mouse oocyte

P4 :

Passage 4

PFA:

Paraformaldehyde

PMSG:

Pregnant mare’s serum gonadotropin

PVDF:

Polyvinylidene difluoride

RT:

Room temperature

SDS:

Sodium dodecyl sulphate

TX:

Triton X-100

WB:

Western blot

ZP:

Zona pellucida

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Author information

Correspondence to Alp Can.

Ethics declarations

The experimental protocol regarding the mouse oocytes and embryos was approved by the IRB (approval number 792/20-18.10.27). All experiments were carried out in accordance with relevant regulations and guidelines of Ankara University Animal Care and Use Committee. Fresh umbilical cords (n = 3) were obtained from full-term female infants after cesarean section succeeding the acquisition of informed consent from the mother (Local Ethics Committee, IRB approval number 18-578-12, 2012) that the experiments conformed to the principles set out in the World Medical Association Declaration of Helsinki.

Conflict of interest

The authors declare that they have no conflict of interest.

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Electronic supplementary material

ESM 1

mEmbryos. Seven movie files are presented showing 20-min long time-lapse movies (40x) before and during the fixation with PFA and PFA/TX groups. Additional 5 min-TX incubation is shown in the PFA+TX and Gly+TX groups; (n=12 independent experiment). Scale bars: 50 μm. (ZIP 28682 kb).

ESM 2

hUC-MSCs. Seven movie files are presented showing 15-min long time-lapse movies (40x) before and during the fixation with PFA and PFA/TX groups. Additional 5 min-TX incubation is shown in PFA+TX and Gly+TX groups; (n=13 independent experiment). Scale bars: 50 μm. (ZIP 37534 kb).

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Cite this article

Celikkan, F.T., Mungan, C., Sucu, M. et al. PFA is superior to glyoxal in preserving oocyte, embryo, and stem cell proteins evidenced by super-resolution microscopical surveys of epitopes. J Assist Reprod Genet (2020) doi:10.1007/s10815-019-01666-9

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Keywords

  • Aldehyde fixative
  • Embryo
  • Fixation
  • Glyoxal
  • Oocyte
  • Paraformaldehyde