## 3 Results
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{
\begin{tabular}{c} **Experimental** \\ **Group M** \\ \end{tabular} } & \multirow{2}{*}{**Samples**} & \multicolumn{2}{c}{**Time in**} & \multicolumn{2}{c}{**Time in**} & \multicolumn{2}{c}{**Staining Result**} \\  & & **Formic Acid** & **EOH** & **Blue (min)** & **Result** \\ \hline \multirow{4}{*}{5} & 5a & 5 & 15 & 5 & Poor \\  & 5b & 5 & 15 & 10 & Poor \\  & 5c & 5 & 15 & 20 & Good \\  & 5d & 5 & 15 & 30 & Good \\  & 5e & 5 & 15 & 40 & Overstained \\ \hline \multirow{4}{*}{6} & 6a & 5 & 0 & 5 & Unstained \\  & 6b & 5 & 0 & 10 & Poor \\  & 6c & 5 & 0 & 20 & Poor \\  & 6d & 5 & 0 & 30 & Unclear \\  & 6e & 5 & 0 & 40 & Unclear \\ \hline \multirow{4}{*}{7} & 7a & 0 & 15 & 5 & Unstained \\  & 7b & 0 & 15 & 10 & Unstained \\  & 7c & 0 & 15 & 20 & Poor \\  & 7d & 0 & 15 & 30 & Poor \\  & 7e & 0 & 15 & 40 & Good \\ \hline \multirow{4}{*}{8} & 8a & 0 & 20 & 5 & Poor \\  & 8b & 0 & 20 & 10 & Good \\  & 8c & 0 & 20 & 20 & Good \\  & 8d & 0 & 20 & 30 & Best \\  & 8e & 0 & 20 & 40 & Overstained \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Results of toluidine blue staining (without the differentiation and dehydration stages). in the dye for 20, 30, and 40 min demonstrated relatively good results. In the second experimental group, the staining was pale and unclear. Similar results were obtained in Group 3 and Group 4, except for the samples stained for 30 and 40 min after pre-immersion in alcohol for 20 min. Table 4 presents the results of the conducted experiments that underwent a stage of differentiation and subsequent dehydration in alcohol solutions of increasing concentration. The staining intensity was evaluated according to the varying staining duration. Staining for 5 and 10 min resulted in pale and uneven histological images in all groups, while a longer duration (40 min) caused overstained and dark images. Figure 1 displays photomicrographs of the experimental groups 1-4. Experiments without the differentiation and dehydration stages were conducted for each group. The excess dye was removed by quickly immersing the slides in distilled water. Samples were dried for 24 h at room temperature (without pre-dehydration in alcohol), cleared in xylene, and coverslipped with BioMount DPX (Biognost, Croatia). In Group 5, good staining results were obtained when the staining duration was 20 and 30 min, while staining for 40 min resulted in overstaining (too-dark images). In Group 6, poor and unclear staining results were obtained after etching the samples with formic acid without subsequent immersion in alcohol. The bone tissue remained unstained after staining for 5 min, while the staining durations of 10 and 20 min resulted in pale images. Increasing the staining duration in the group resulted in unclear images. For the  
Figure 1: Photomicrographs of cross-sections through rabbit tibia (magnification 20\(\times\)). The images present the results from the conducted experiments with differentiation and dehydration stages (experimental groups 1-4 and their subgroups a–e, respectively). The results are marked as: “poor” (**1a,1b,2b,2c,3c,3d,4b,4c**); “unstained” (**2a,3a,3b,4a**); “unclear” (**2d,2e,3e**); and “good” (**1c–1e,4d,4e**). samples in experimental group 7, good results were obtained only after a longer staining duration--40 min. On the other hand, the samples from Group 8 demonstrated good staining results. Although immersion in the dye for 5 min led to pale staining, the results improved significantly when the staining time increased. The best results were obtained after a staining duration of 30 min. Further increasing the time led to the overstaining of the samples (Table 5). Figure 2 displays photomicrographs of the experimental groups 5-8. In an attempt to optimize the staining protocol, we introduced a stage of washing the samples in distilled water after the immersion in ethanol, and under these conditions, more uniform staining was observed.