*4.3. Morphometric and Immunofluorescent Analysis of the Brain*

For histological analysis, rats were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg) and intracardially perfused with 4% paraformaldehyde (PFA, Sigma) in phosphate-buffered saline (PBS, pH 7.4). The brains of the rats were isolated from the skull, post-fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose. Frozen frontal sections of the brain were obtained through the epicenter of ischemic injury using a cryostat (Microm HM 560, Thermo Scientific, Waltham, MA, USA).

Morphometric analysis of the volume of the cerebral infarction included macroscopic evaluation of the infarct area of the whole brain by capturing digital images and microscopic study every 10 frontal brain sections (with 200 μm interval) through the ischemic injury area, after staining with hematoxylin and eosin. To calculate the infarction volume, maximal depth and maximal diameter of the infarct cavities were estimated using ImageJ software (NIH). The calculation was carried out as V = 1/3 × maximal depth × π × (maximal diameter/2)**2**.

Immunofluorescent staining was performed on 20 μm sections of frontal brain using primary antibodies (Ab) reacting with cell specific markers (Table 2). The viability of neural cells was evaluated using Ab to pro-apoptotic protein (Caspase3) and heat shock protein 70 kDa (Hsp70). Synaptic function of preserved neurons was assessed with Ab against synaptophysin and postsynaptic density protein 95 kDa (PSD95). Responses of glial cells were assessed with Ab to GFAP for astrocytes, Ab to Olig2 for oligodendrocytes and Ab to Iba1 for microglia. Anti-human nuclear antigen (HNA) antibodies were used for identification of human UCBC in the rat brain. Double immunofluorescent staining with Ab to HNA and Ab to human VEGF, GDNF, and NCAM were employed to analyze the expression of those recombinant molecules in UCB-MC. For immunofluorescent labelling, sections were incubated with secondary antibodies (Table 2). Appropriate secondary Alexa Fluor 647 and 488 conjugated Abs were used for immunofluorescent labelling of the target molecules. Cell nuclear counterstaining was performed with DAPI (10 μg/mL in PBS, Sigma), sections were embedded in glycerol (GalenoPharm, Saint Petersburg, Russia) and observed under a LEICA TCS SP5 MP microscope (Leica Microsystems, Wetzlar, Germany).

Digital images were captured using identical confocal settings in areas of 0.05 mm<sup>2</sup> and analyzed with ImageJ software (NIH) in 10 captured fields in the peri-infarct zone (Figure 2C) in each section, as described previously [25]. The number of immunopositive cells for apoptosis protein Caspase3 and oligodendroglial cells transcription factor Olig2 were counted in regard to nuclear counterstaining with DAPI. Neuroglial markers for astrocytes (GFAP) and microglia (Iba1) were evaluated as the immunopositive areas and presented in percentages. The level of synaptic proteins (synaptophysin and PSD95) and Hsp70 immunoexpression in brain sections was evaluated as mean pixel intensities.


**Table 2.** Antibodies used in immunofluorescent analysis.
