The goal of this protocol is to describe how to assess the quantity and the distribution of iron deposition in the brains of an AD mouse model. In this protocol, we use eight-months-old 5xFAD transgenic mice as the AD mouse model and compared them with wild-type mice of the same age. We include details of procedures for preparing the chemical reagent, sectioning the brain, performing the Perls/DAB staining, and analyzing the resulting images.
Anesthetize the mouse properly and check the depth of anesthesia and analgesia. Expose its heart, then cut open the right atrium. Inject 20 milliliter PBS and 4%PFA from the left ventricle in sequence.
Notice that fixation tremors should be observed. Decapitate the mouse and extract its brain. Then, fix the brain in 4%PFA for 12 to 24 hours.
Immerse the brain into 15%and 30%sucrose for 12 to 24 hours. Cut the brain sagittally from the middle and use either half for sectioning. Mount the brain on the knob, put a tin foil ring around the brain, and add OCT into it to embed the brain.
Set the thickness of sections and temperature of cryostat. Then, cut the brain. If the sections fold, use soft brushes to unfold the sections.
Collect each section to the slides with PBS, then eliminate the bubbles on the section. Select a slide with intact brain tissue and place it in a plastic staining box filled with PBS. Position the box on rotary shaker set to a slow speed for five minutes to thoroughly rinse off residual OCT compound.
Prepare 20 milliliter of 2%potassium ferrocyanide and equal volume of 2%hydrochloric acid. Then, mix them in a 50 milliliter centrifuge tube. Secure the slide in the tube using forceps and incubated the mixture in preheated water bath at 60 Celsius for 30 minutes.
Wash the slide with PBS and wipe off excess liquid using tissue paper. Lay the slide flat on the lab bench and even apply DAB solution over the tissue using a pipette. Incubate for 10 minutes to enhance Perls staining.
Remove the excessive DAB solution and rinse the tissues by washing three times with PBS. Dehydrate the section sequentially in graded alcohol solutions and xylene for three minutes each. Cover the sections with a neutral gum and a cover slip.
Then, allow them to dry in a fume hood. Turn on the microscope. Adjust the brightness of the light source.
Focus on the brain section under a 4X magnification objective. Move the microscope stage to focus on the areas with high Perls/DAB staining signals, especially the hippocampus and the cerebral cortex. and capture images.
Open the objective image at 10 magnification, and then convert the image format to 8-bit grayscale. The gray values of the image were converted to OD values, and then the threshold function was used to cover the Perls/DAB staining-positive areas. Select the following setup options and most importantly, select limit to threshold to exclude background noise.
Finally, select measurements to obtain results for statistical analysis. To investigate the distribution and accumulation of iron in an AD mouse model, we performed Perls/DAB staining of sagittal brain sections. High Perls/DAB signals were observed in the hippocampus and cortex, especially in the subiculum of the hippocampus of 5xFAD mice, whereas the brains of both two month and eight month wild-type mice showed weaker signal.
Under 40 plus magnification, the signal in 5xFAD mice appears in A-beta plaque-like structures, consistent with previous studies. These results demonstrate the effectiveness of Perls/DAB standing as a histochemical technique for iron detection. We also show two instances of failed staining.
Excessive covering or inappropriate dehydration will result in these cases. Perls/DAB staining provides a stronger signal and better background contrast than conventional Perls staining, making iron detection more sensitive and accurate. In addition, it detects the ferric iron in loosely bound protein complexes.
Iron that is strongly bound, as in hemoglobin, will not react. This is greatly reduces unwanted signals caused by iron in red blood cells and hemoglobin. Overall, Perls/DAB staining is appropriate for animal experiments and the pathological investigations that require medial specificity and sensitivity.
It provides researchers with a method to visualize and quantify iron accumulation at the expense of less time and cost.
