Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

All experimental procedures are approved by the Sacred Heart University Institutional Animal Care and Use Committee and are in accordance with the NIH Guide for the Care and Use of Animals.

1. Isolation and infiltration of brain tissue

1. Premix solutions A and B of the Golgi staining kit 24 h prior to use and keep in dark bottles and/or in dark. Make approximately 80 mL of solution A and B mix which is sufficient to change the solution after 24 h. Store in airtight bottles.
   NOTE: Perfusion with either saline or paraformaldehyde is not necessary.
2. Sacrifice rats by guillotine following carbon dioxide euthanasia and remove brains within seconds. Rinse brains in saline if required but is not necessary.
   1. Place the brain, cortex down so that the hypothalamus is visible because the cuts are made anterior and posterior to it, on a non-porous surface. Cut into an anterior (contains the prefrontal cortex) and posterior (contains the hippocampus) block as shown in Figure 1.
3. Place blocks into the premixed solutions of A and B, making sure that they are well immersed in the solution. Ensure that the volume of solutions A and B is sufficient to immerse the blocks. Store blocks in either brown bottles or clear bottles covered with foil (to keep light out) in the dark at room temperature.
4. Replace solution after 24 h and keep in the dark for another 13 days at room temperature.
   NOTE: If used with mouse brains, there is no need to block, and the entire brain can be placed in the solution. If staining brain areas, which are different in size, the time in solutions A and B may have to be determined by trial and error.
5. After two weeks the tissue is infiltrated well with solutions A and B, transfer the blocks to the cryoprotectant solution (solution C in the Golgi staining kit), and leave them for 48-72 h at 4 °C.
   NOTE: After cryoprotection, blocks may be frozen until further processing. Also, note that all solutions from the kit are collected and disposed of as hazardous waste.
6. Freeze brain, cortex down, on a glass slide on dry ice. Once frozen either cut on the cryostat or store at -80 °C until sectioning using a cryostat.
   ​NOTE: Do not freeze in liquid nitrogen as this produces cracks in the tissue.

2. Sectioning of brain tissues

1. Place a small amount of tissue medium on a pre-cooled cryostat chuck. Mount blocks on cryostat chucks by thawing one side of the block slightly in hand (gloved) and placing it on the tissue medium.
   NOTE: It is not necessary to embed the tissue. The block containing the hippocampus is somewhat easier to cut than the block with the prefrontal cortex because the latter is anterior to the corpus callosum and the two halves are separate.
2. Cut 100 µm sections on a cryostat at -22 °C and mount onto subbed slides. Sections up to 150 µm of thickness can be used. Try to mount 3-4 coronal sections per slide as this decreases the number of slides that require processing. Use one of the following techniques to keep the sections frozen till they get on the slide.
   NOTE: These sections are not fixed in the usual way and therefore they tend to melt quickly. Allow the sections to melt on the slide. If they begin to melt before being placed on the slide, it is impossible to get them to be flat on the slide. There are several techniques to do this.
   1. Use a freezing spray on the knife and the block. Depending on the temperature and humidity in the room this may be necessary for every section. Use either the antiroll plate or a brush to keep the section flat while cutting.
      NOTE: Make sure to have enough freezing spray. Several cans can be used when cutting 20 blocks.
   2. Thaw mount, if possible, by using a room temperature slide and quickly appose it to the section. With practice, one can mount several sections at once. If this doesn't work, keep slides in the cryostat so they are very cold and transfer the section with cold forceps or a cold paintbrush and then thaw mount.
3. Clean the knife with paper wipes between slices. If the knife requires more cleaning, use 100% ethanol (EtOH), and let it dry before cutting the next slice.
4. Once mounted onto the slide, place the slide flat on cardboard slide trays and allow sections to dry at room temperature (for several hours to a maximum of 48 h) in the dark. Do not cover the slide tray. Ensure that the sections are completely dry before Golgi impregnation. Store flat, in the dark, on slide trays until Golgi impregnation.
   ​NOTE: Drying the sections does not cause any damage.

3. Staining and dehydration of brain tissue

1. Perform staining in glass staining dishes. Mix solutions D and E immediately prior to staining. As per the protocol, add one part of solution D, one part of solution E, and two parts of distilled water. For glass staining dishes, make a 200 mL solution to completely submerge the sections. For Koplin jars, this requires less volume.
2. Place slides in racks spaced far enough apart to allow solution access to sections.
3. Place sections in distilled water for 4 min (2x) before placing them into the Golgi impregnation staining solution for 10 min. Change the staining solution every 70 sections. Change the distilled water when it turns yellow.
   NOTE: The timing for the staining step is critical. Too short does not allow enough staining and too long causes over staining, making the dendrites hard to separate when doing analysis. Once out of the staining solution the timing is less critical.
4. Dehydrate the sections as follows: 70% EtOH (5 min), 95% EtOH (5 min; 2x), 100% EtOH (5 min; 2x), clearing agent (5 min; 3x). Change all solutions often, especially the 100% EtOH and clearing agent to ensure the sections remain anhydrous.
   NOTE: It is not necessary to go through a 50% EtOH step. Counterstaining is not needed for cell visualization because the Golgi impregnation provides sufficient contrast. The use of other clearing agents has not worked as well as the one used here (see Table of materials).
5. Coverslip with glass coverslips that are 60 mm long with a generous amount of mounting medium. Make sure that there are, as few air bubbles as possible. If needed, carefully remove, and redo the coverslip (can be done even weeks later). Do this very slowly in order not to damage the section (can be done because of the large amount of mounting medium).
   NOTE: A large amount of mounting medium is somewhat messy but still necessary because enough mounting medium must be used to cover the thick 100 µm sections.
6. To ensure that only one coverslip is placed on the sections, separate the coverslips in advance of the process.
7. Once cover slipped, dry slides flat on any non-porous paper for 3-5 days, moving them slightly, especially after the first day, to avoid sticking. After 3-5 days transfer the slides to slide holders and, ideally, dry slides for at least 3 weeks before examining them. Keep slides flat to decrease the possibility of air bubbles forming.

4. Determination of dendritic spine density

1. For analysis of dendritic spine density in pyramidal neurons of both the mPFC and the CA1 region of the hippocampus, examine the most lateral secondary basal dendrites and the most lateral tertiary apical dendrites as described in step 4.1.1 (Figure 2).
   1. Choose a dendrite, measure the length of the dendrite using an image analysis program, count the spines on the dendrites using a hand counter, and record both length and number of spines.
2. Study and analyze six cells per region (mPFC, CA1) per brain. Quantify a minimum of six brains per group as previously described^7,8. Choose neurons that meet the following criteria for analysis: cell bodies and dendrites are well impregnated; dendrites are distinguishable from adjacent cells and are continuous.
3. Count the spines at 1000x (oil-immersion) by hand counting with a light microscope and measure dendritic length using an image analysis program. Calculate spine density by dividing the spine number by the length of the dendrite and express data as the number of spines/ 10 µm dendrite.
   NOTE: There are far more sophisticated methods for differentiating spine subtypes and dendritic architecture that can be used, but hand counting with a light microscope at 1000x can give a rapid result that can then determine if further investigation is necessary. Although every effort is made to consistently sample similar dendrites, there are variations in thickness that may affect the counting.