*4.7. Immunofluorescence/Immunohistochemistry*

To investigate the expression of the proteins of interest, we used immunofluorescent labeling in combination with DAPI nuclear counterstaining or immunohistological staining visualized with 3,3 -diaminobenzidine (DAB) chromogen (Dako).

After deparaffinization and rehydration of slides, antigen retrieval was achieved by heat-induced epitope retrieval procedure in the citric acid buffer (10 mM, pH 6.0). Nonspecific binding sites were blocked with Tris-buffered saline (TBS) containing 5% bovine serum albumin (BSA) and 0.025% Triton X-100. Slides were incubated overnight at 4 ◦C with primary antibodies, as listed in Table 1.



Abbreviations: Iba1, ionized calcium-binding adapter molecule 1; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; NfL, neurofilament light chain; SYP, synaptophysin.

For the sections immunolabeled and visualized by using the DAB chromogen, the brain slices were incubated with an appropriate biotinylated secondary antibody (Table 1), diluted in the antibody solution buffer for 1 h at RT, followed by the streptavidin–HRP conjugate for 30 min at RT. Following the application of DAB, reaction with HRP produced a brown precipitate. The slides were then dehydrated, immersed in xylene, and mounted. For the immunofluorescence labeling, appropriate fluorochrome-conjugated secondary antibodies were applied for 1 h at RT. Cell nuclei were counterstained with DAPI, and the slides were mounted in anti-fade mounting medium.

Immunolabeled sections were examined by light or epifluorescence microscopy (Olympus BX 51 microscope with Olympus DP 70 digital camera, Olympus, Tokyo, Japan).

Quantification of neurodegeneration and the glial response in the OTs was done on Fluoro-Jade C-stained and Iba1- or GFAP-immunolabeled coronal sections cut in the range of −1.34 to−2.30 from bregma [85] using ImageJ software (NIH, Bethesda, Md, USA). Quantification of microgliosis and astrocytosis was made by measuring the percentage (%) of the Iba1- or GFAP-immunoreactive areas. Microphotographs of two sections from each animal, at ×400 magnification, were transformed to 8 bit images and auto-thresholded (0 being white and 255 being black), which enabled differentiating positive immunoreactions from the background and calculating the immunoreactive area fraction. A region of interest (ROI) was drawn around the OT. The area fractions were averaged for each animal and each experimental group.

In the OTs, quantification of the DAB staining intensity was also done with ImageJ software. Briefly, mean gray values were collected from the ROIs selected in the images of the OTs, and optical density (OD) was calculated with the following formula: OD = log(max gray intensity/mean gray intensity).

In the LGN and the SC, we evaluated the intensity of SYP immunofluorescent staining. Conditions of the microscopy and photography were maintained constant throughout the experiment, and immunoreactivity was quantified by measuring the integrated optical density. In the mentioned nuclei, we also evaluated the microglial response by counting the number of Iba1-stained cells in the immunofluorescently labeled sections.
