Data on morphology, large-scale chromatin configuration and the occurrence of proteins and rRNA in nucleolus-like bodies of fully-grown mouse oocytes in different fixatives

Here we provide data on accessibility of nucleolus-like bodies (NLBs) of fully-grown (GV) mouse oocytes to fluorescence in situ hybridization (FISH) probes and anti-nucleolar antibodies as well as on oocyte general morphology and large scale chromatin configuration, which relate to the research article “High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes” (Shishova et al., 2015 [1]). Experimental factors include: a cross-linking reagent formaldehyde and two denaturing fixatives, such as 70% ethanol and a mixture of absolute methanol and glacial acetic acid (3:1, v/v).


Value of the data
These data demonstrate that the occurrence of nucleolar proteins and RNAs in NLBs should be examined not only after oocyte fixation with paraformaldehyde but also after their fixation with 70% ethanol.
These data are valuable to researchers interested in investigating the molecular composition of NLBs in mammalian oocytes.

Data
Cross-linking (formaldehyde) and denaturing (70% ethanol and methanol/glacial acidic acid, 3:1, v/ v) fixatives exert different effects on oocyte and NLB morphology (Fig. 1a, a 0 , a″), large-scale chromatin configuration (b, b 0 , b″) and on accessibility of the NLB material to immunofluorescence (c, c 0 , c″) and fluorescence in situ hybridization (FISH) probes (Fig. 2). Fixation with paraformaldehyde (PFA) best preserves the oocyte phenotype ( Fig. 1a) and chromatin configuration (b), but it does not permit to label the nucleolar protein NPM1 (c) and rRNAs (Fig. 2a-c) inside NLBs. In the NLB mass, rRNAs became accessible to different FISH probes [1] only after oocyte fixation with 70% ethanol (Fig. 2a 0 or with a mixture of methanol and acidic acid (2a″-c″) despite the mixture can deteriorate resolution labeling as compared with that in the ethanol-fixed oocytes (a 0 , a″) ( Table 1).

Oocyte collection and fixation
Fully-grown oocytes were collected from C57Bl/6 mice aged 4-8 weeks following the standard hormone administration with PMSG as described in [1]. Oocytes were fixed either with freshly made 3% PFA in PBS (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , and 8.1 mM Na 2 HPO 4 , рН 7.2) or with 70% ethanol in bidistilled water or with a mixture of absolute methanol and glacial acetic acid (3:1, v/v). In all cases, the fixation procedure continued for 20-25 min at room temperature. PFA-fixed oocytes were then treated with 0.5% Triton X-100 in PBS for 10 min, and the other oocytes were exposed to 0.2% Triton X-100 for 5 min to increase accessibility of the used probes and antibodies to target biomolecules.

Phase contrast
Oocytes were fixed with either of three fixatives, placed in PBS and examined as described in Section 2.6.
(Russia), conjugated with Cy3 at the 5 0 -terminal end and had the stock concentration about 2 μg/μl.
After DNA-staining, IF and FISH oocytes were mounted in Vectashield s (Vector Laboratories, USA), and examined under a confocal microscope within the next one-two days.

Image acquisition
Eight-bit digital images of oocytes were acquired with a DuoScanMeta LSM510 confocal laser scanning microscope (Carl Zeiss, Germany) equipped with a Plan-Apochromat 63 Â /1.40 (numerical aperture) oil Ph3 objective.

Acknowledgments
The study was supported by the Russian Scientific Foundation (Grant no. 14-14-00856).

Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2016.03.085.