# Quantitative Immunofluorescence Mapping of HSP70's Neuroprotective Effects in FUS-ALS Mouse Models
## 3 Results  
Mice of FUS [1-359], FUS [1-359]/HSP70 (in), and FUS [1-359]/HSP70 (out) populations, not randomized to the experiment, lived for 14-27 weeks since birth on average, whereas the populations of all single HSP70 mice and wild-type animals lived much more than 30 weeks (Figure 2). In FUS [1-359]/HSP70 (in) mice, the overall survival is greatly higher than in FUS [1-359] mice, but even a lower duration of overall survival was shown for FUS [1-359]/HSP70 (out) mice. However, all the FUS-expressing mice were symptomatic for muscle atrophy independently of their survival terms. Figure 2: Overall survival for animals with aberrant FUS expression and FUS/HSP70 overexpression. The plot shows the overall survival of animals with human FUS [1-359] gene insertion with the ratio of mice staying alive at certain week since birth. Censored values reveal the rate of mice staying alive in each group to the moment of immunofluorescence study. Figure 3A shows images of the primary motor cortex stained by anti-NeuN antibodies for mice from six groups with the corresponding boxplots at Figure 3B,C. The weakest level of fluorochrome luminance is found in the primary motor cortex of FUS [1-359] mice. At Figure 3B, the boxplot illustrates a significant and considerable decrease in average mean value for the number of NeuN-positive cells in FUS [1-359] animals compared to both groups of single transgenic HSP70-expressing mice and controls, whereas double transgenic mice have no statistical difference of the value from all the groups except FUS [1-359] animals. The differences at the NeuN/DAPI ratio calculations are less prominent between the groups. FUS [1-359] mice show a lower ratio of NeuN-positive to DAPI-stained cells compared to all animals lacking aberrant FUS expression (Figure 3C), although no increase occurs in double transgenic animals compared to FUS [1-359] mice. The distribution for  
Figure 3: Immunofluorescent study. NeuN expression in the primary motor cortex of double FUS [1-359]/HSP70 transgenic mice compared to single FUS [1-359] transgenic animals and control groups (\(n=6\) for each group). **(A)**. Microphotographs of cerebral sections, d = 5 \(\upmu\)m., ob. 10. Scale bar size: 20 \(\upmu\)m. Shades of red color mark the primary antibody (NeuN) used, blue color marks DAPI counterstainedaining. **(B,C)** Boxplots of statistical data: names of study groups are given at X axis, parameters measured—at Y axis. *—statistical difference is significant at \(p<0.01\) (\(p<0.0001\) for NeuN, \(p=0.0002\) for NeuN/DAPI). NeuN-stained cells is normal, and for the NeuN/DAPI ratio is not, so not the average means but general samples are compared for the NeuN/DAPI ratio. The number of GFAP-positive cells has almost no difference between the groups, excluding FUS [1-359]/HSP70 (out) animals with a slight statistically significant increase in GFAP + cells compared to HSP70 (in), their counterparts with intracellular HSPA1A accumulation, as well as with FUS [1-359] mice (Figure 4A,C). However, Figure 3B and the relevant graph at Figure 4D reveal a colossal increase in number of S100\(\emptyset\)-positive cells in all groups expressing FUS compared to control wild-type animals, what is well shown by the dynamics of fluorescent pattern at Figure 3B. Single FUS [1-359] transgenic animals also have a significant difference in the number of S100\(\emptyset\)-positive cells to HSP70 (out) group, whereas this pattern is not seen for anti-GFAP staining. Figure 5A shows visual differences between the groups for Iba1 antibody staining. Statistically significant differences are visible after calculations, represented at the corresponding boxplot (Figure 5C). FUS [1-359] animals have less Iba1-positive cells than double transgenic mice with intracellular HSPA1A expression, FUS [1-359]/HSP70 (in), as well as with the animals of HSP70 (in) group. Further, we observe control animals to have less Iba1 expression than all the groups with HSPA1A overexpression. Figure 4: Immunofluorescent study.