2.6. Tissue Processing for Histology

About 2–3 mm tissue was trimmed and processed for paraffin embedding. Briefly, the tissue was dehydrated with 70%, 80% alcohol, followed by 95% ethanol and 5% methanol for 1 h each. Then three changes in absolute alcohol, 1 h each, followed by two changes in xylene (45 min each). The tissue was embedded with paraffin at 56 °C for two changes, 1 h each, followed by another 4 h embedding with fresh paraffin solution. The paraffin block was made and a 5 μm thick section was cut using a rotary microtome (Microm, HM-310, Charleston, SC, USA) and placed on a poly-L-lysine-coated glass slide.

The paraffin-embedded sections were stained with hematoxylin-eosin (H&E). Briefly, the sections were deparaffinized by dipping in xylene, two times, 5 min each, followed by immersion in consecutive 100%, 90%, 70% and 50% solution of alcohol for 1 min each and then immersed in tap water for another 5–10 min. Then the sections were stained with Mayer’s hematoxylin solution for 5 min, followed by an immersion in tap water for 10 min. The tissue was treated with a bluing solution for 1 min, followed by a wash in tap water for another 5 min. Then the tissue was decolorized with 1% acid alcohol (1:1) and dipped in tap water for another 5 min. The tissue was then treated with 70% alcohol and counter-stained with eosin-Y for 2 min, and washed with 70% alcohol for two times, 1 min each. Then the sections were dehydrated with graded alcohol and cleared with xylene (5 min each, two times) and mounted using DePex mounting media. Images were taken using a compound light microscope (Olympus, Japan) using 10x and 40x objectives.

DNA fragmented cells were labeled by TUNEL Assay Kit-BrdU-Red as described, previously [32,33]. Briefly, paraffin-embedded liver, kidney, spleen and lung tissues were sectioned at 5 μm thickness using rotary microtome and taken on a poly-L-lysine-coated glass slides. The sections were deparaffinized and rehydrated and then treated with 0.85% sodium chloride solution for 5 min, followed by three washes with PBS (0.1 M, pH 7.4), 5 min each. Then the sections were treated with proteinase K (20 ng/μL) for 5 min, followed by three washes with PBS, 5 min each. The sections were then incubated with BrdU solution for 1 h at 37 °C in a humidified chamber in the dark, followed by washing three times with PBS, 5 min each. Then the sections were incubated with anti-BrdU antibody conjugated with Texas-red, followed by three washes with PBS, 5 min each. All sections were counterstained with DAPI (20 mmol/L) for 10 min at room temperature in the dark and washed thoroughly with distilled water before being mounted with an anti-fading medium. The images were captured using fluorescent microscopes (Leica, Germany) by 40x objectives (total magnification 400×) with appropriate excitation/emission filters (ex/em: 488/576), so that the TUNEL-positive cells produced red-fluorescence. The number of TUNEL-positive cells were counted manually in each microscopic image and expressed as the number of TUNEL positive cell/microscopic fields. At least 30–40 images were taken from three mice of each group to obtain a mean value. The counting was performed independently by two researchers, who were blind to the group identity of the sample.

The CR staining was performed to label the amyloid deposits in the peripheral organs, especially in the spleen and in the liver with some modifications [23]. Briefly, the paraffin embedded sections (5 μm thick) were deparaffinized, and rehydrated with graded alcohol solution and then washed with lukewarm tap water for 20–30 min. The sections were stained with 0.5% CR solution, dissolved in 80% ethanol along with 1% NaOH for 1 h at room temperature. Then the sections were rinsed in running, lukewarm tap water for 15–20 min, followed by counterstaining with Mayer’s hematoxylin solution for 5 min and washed three times with distilled water, dehydrated through an ascending alcohol series (70%, 90%, and 100%), and then placed into xylene and cover-slipped with DePeX. The sections were visualized using a compound light microscope (Olympus, Japan) using 40× objectives (total magnification = 400×) with a radish pink color indicating amyloid deposition.

For DAPI and PI staining, the paraffin-embedded sections were deparaffinized and rehydrated with a graded alcohol series (70%, 90%, and 100%) and then stained with DAPI (20 mmol/L) for 10 min at room temperature in the dark. For propidium iodide (PI) staining, the sections were stained with 500 nM concentration of PI for 5 min at room temperature, in dark, after which the sections were washed with distilled water for tree time, 5 min each, and mounted with aqueous anti-fading mounting media. The images were captured by a fluorescent microscope (Leica, Wetzlar, Germany) using the appropriate excitation/emission filters. The fluorescent intensity of DAPI and PI stained images were measured manually using ImageJ software and expressed as fluorescent intensity in arbitrary units (AU).

The immunohistochemistry protocol for pTau was described elsewhere [23,24,25,26,27,28]. Briefly, the paraffin sections were first deparaffinized and rehydrated with graded alcohol. Then the sections were washed with PBS-Triton-X-100 (0.5%, PBS-T) for 5 min and blocked with 10% goat serum (dissolved in PBS-T) for 30 min at room temperature with gentle shaking. The sections were incubated with pTau antibody (mouse monoclonal, 1:200), overnight on the shaker. On the next day, the sections were washed three times, 5 min each, and incubated with anti-mouse secondary antibody, conjugated with Alexa-488, for 1 h at room temperature with gentle shaking. Then the sections were washed with water, three times, 5 min each, followed by counterstaining with PI (500 nM) for 5 min. Finally, the sections were washed, and mounted with anti-fading mounting media and the images were taken using fluorescent microscopes (Leica, Wetzlar, Germany) with appropriate excitation and emission filters. The bright green signal was considered as pTau-positive cells. The number of pTau-positive cells were counted manually from the spleen tissue of three mice in each group with at least 15–20 randomly captured images and expressed as mean pTau positive cells/microscopic field.

The detailed protocol for immunoperoxidase techniques for caspase-3 was followed from the previously described protocol with some modifications [25]. Briefly, the paraffin sections of liver, kidney, spleen and lungs were deparaffinized, and rehydrated. The sections were then incubated with 3% hydrogen peroxide solution for 30 min at room temperature to inhibit endogenous peroxidase. Then the sections were rinsed with PBS, three times, 5 min each, and blocked with 10% normal goat serum (NGS), and underwent gentle shaking for 1 h at room temperature. The tissue sections were then incubated with cleaved caspase-3 antibody (rabbit, monoclonal, 1:200) with gentle shaking overnight at 4 °C. On the next day, the sections were thoroughly washed 3 times, for 5 min each, and incubated with a biotinylated anti-rabbit secondary antibody (1:250) for 1 h at room temperature. Finally, the sections were incubated with a peroxidase substrate solution, supplied with the ABC kit (Vector Laboratory, Burlingame, CA, USA), and the signal was developed using diaminobenzidine (DAB) until the desired staining intensity emerged. The sections were counterstained with hematoxylin for 10 min and then washed, dehydrated with graded alcohol, cleared with xylene, mounted on slides, cover slipped with DePex, air dried, and visualized using a compound light microscope (Olympus, Japan) with 40x objectives (total magnification 200×).

Detailed protocol for Western blot techniques was described previously [24]. Briefly, about 100 mg of flash-frozen spleen tissue from WT, 3xTg, 5xFAD and the 3xTg or 5xFAD mice treated with CGR were lysed with cold radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors. The total protein was measured by the bicinchoninic acid assay (BCA assay) and about 200 μg of protein from each group was loaded on 4–20% Tris-glycine gel and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) membranes and the membranes were probed with different primary antibodies (1:1000), such as caspase-3, 6, Bcl₂, TNF-α, IL-1β, IL-10, pTau, pAkt and pGSK-3β followed by their respective secondary antibodies (1:10,000). The blots were developed with SuperSignal™ West Femto Maximum Sensitivity Substrate using gel documentation system (Bio-rad). The relative optical density was measured using Image-J software (https://imagej.nih.gov/ij/) (accessed on 2 April 2021). To ensure equal protein loading in each lane, the blots were re-probed for GAPDH antibodies.

The data were analyzed using the one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) post hoc test. All the data were expressed as mean ± SEM and the p ≤ 0.05 was considered as significant.