Add 2.4 g Mowiol 4–88 (Hoechst) or Gelvatol 20–30 (Monsanto) to 6 g glycerol in a 50-ml conical tube. Stir to mix.

While stirring, add 6 ml distilled water and leave for 2 h at RT on a rocker.

Add 12 ml 0.2 M Tris (pH 8.5).

Add NaN₃ to a final concentration of 0.02% (optional).

Incubate the tube at 50°C for 10 min or until Mowiol is dissolved.

Clarify by centrifugation at 5,000 g for 15 min. Store 1-ml aliquots in microcentrifuge tubes at −20°C.

Warm tubes to room temperature for use. Opened tubes can be stored at 4°C for approximately 1 month. Discard if any crystalline material is seen in the tube or on the slides.

For fluorescence, add 1,4-diazobicyclo-(2.2.2)-octane (DABCO) to 2.5% to reduce fading. This is a free radical scavenger. Note: since this is an aqueous medium fluorescence quality can diminish overtime. It is best to image 1 day after tissue is coverslipped.

Heat 10 ml 0.1 M phosphate buffer, pH = 7.4 (PB) to 60°C in a microwave.

In a fume hood, for a 4% solution, add 0.4 g paraformaldehyde. Caution: paraformaldehyde fumes are toxic.

Stir until dissolved. If powder does not dissolve, add a drop of 5 N NaOH.

Chill on ice. Filter and adjust pH to 7.4 if necessary. Keep for about 1 week at 4°C.

3% (v/v) normal horse serum. (Sometimes less background is obtained by substituting normal goat serum.)

0.5% (v/v) Triton X-100.

0.025% (w/v) NaN₃ in phosphate buffer saline (PBS).

Mice are euthanized using an overdose of isoflurane or pentobarbital delivered via intraperitoneal injection.

Eyes are rapidly enucleated with curved scissors (care must be taken to not damage the optic nerve otherwise the retina will pull away from the eyecup during dissection), and rinsed in cold 0.1 M PB with 2 mM Ca²⁺ and Mg²⁺ added.

Eyecups are prepared by cutting just behind the ora serrata followed by removal of the lens. Eye is placed in a Petri dish filled with cold PB (with Ca²⁺ and Mg²⁺). Hold eye in place with forceps and puncture eye with a number 11 scalpel blade just behind the ora serrata. Roll eye onto optic nerve. Use microscissors to cut around the equator. It may be easier to rotate the eye with forceps as you cut. Gently peel the front portion of the eye away from the eyecup. Roll the lens out of the way. Tilt the eyecup on its side to drain the remaining vitreous. Note: Any remaining vitreous will crystallize when frozen and create problems for sectioning. The vitreous freezes at a different rate causing the nerve fiber layer and the ganglion cell layer to be pulled away from the remaining retina. Also the remaining vitreous will fracture during sectioning. This causes the block to cut unevenly. The section will buckle.

Eyecups are transferred (via a cut plastic transfer pipette) to the fixative solution. Tissue is fixed for 5–20 min by immersion in 4% PFA. We use glass vials for fixation to avoid plastics that may leach from other containers. PB is used because PBS has a higher osmolarity that can cause tissue damage and poor tissue preservation.

The fixed eyecups are washed in PB and then cryoprotected at 4°C by sequential immersion in ice cold 10, 20, and 30% (w/v) sucrose in PB. Use a cut plastic transfer pipette to gently move eyecups into each new solution. Allow each eyecup to sink to the bottom of the vial at each step. (About 10–30 min.) The eyecups can sit overnight in the 30% sucrose at 4°C. The eyecups are then immersed into OCT (Sakura Finetek, Torrance, CA). Gently mix to remove the extra 30% sucrose. You should be able to see the mixing of the sucrose and the OCT. If extra sucrose remains inside the eyecup this will also cause sectioning problems. The sucrose will crumble during sectioning and the retina will be pulled along with the sucrose.

The tissue is then embedded in OCT (use plastic molds, such as the cap of a microcentrifuge tube, to help position the eyecups) and quickly frozen by immersion into isopentane/dry ice. Isopentane is placed in a glass beaker. Dry ice is packed around the beaker. Allow for the isopentane to equilibrate (otherwise freeze fracture will occur). The tissue is frozen and equilibrated when bubbles no longer form. The OCT-embedded eyecups can be stored at −80°C, but it is preferable to proceed to the sectioning.

The eyecup is removed from the molds and cut at 12–15 μm on a cryostat at −18 to −21°C (2 h equilibration in the cryostat is best). Optimal cutting temperature is determined by the humidity in the cryostat chamber and section thickness. Sections are collected onto Super-Frost glass slides, air-dried (this helps keep the sections attached to the slide during immunostaining and gently dehydrates the tissue for best morphology), and stored at −80°C until used for staining.

Eyecup sections are thawed to room temperature (RT) and dried. Create a circle around the section with a PAP pen and allow to dry. Block by incubation at RT for 30–60 min in AIS.

Aspirate off solutions to preserve the well created by the PAP pen. Incubate with primary antibody diluted in AIS for either 1–2 h at RT or at 4°C overnight. (Use a humid chamber to prevent the sections from drying. Use only enough antibody solution to cover the section. Otherwise the solution will flow over the well.)

After three quick washes (5–10 min each) in PBS, the sections are incubated for 1 h at RT in the appropriate secondary antibody coupled to either Cy3 or Cy2 (Jackson ImmunoResearch Laboratories, West Grove, PA), or to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen, Carlsbad, CA) diluted 1:500 to 1:2000 in PBS or AIS. (Keep sections protected from light beyond this step.)

The slides are washed again three times in PBS (5–10 min each) with one rinse with water (to reduce salt crystal formation) and then coverslipped with AMM. The coverslipped slides are left in the dark overnight to harden before oil immersion lenses are used. (Nail polish is used to seal the edges of the coverslip.)

Slides are viewed on a fluorescence or confocal laser scanning microscope using 40× or 60× oil immersion objectives.

Following dissection of a mouse retina, four radial cuts are made to flatten the tissue.

The flattened retina is placed on a piece of filter paper to prevent the retina from folding or rolling up.

The retina on the filter paper is fixed for 1–30 min by immersion in 4% PFA, and then washed in PBS.

The tissue is gently removed from the filter paper (this becomes easy after fixation) with a fine paintbrush and is then incubated with primary antibody diluted in AIS for 1–3 days at 4°C on a gently rocking (or orbital) platform.

After three 1-h washes in cold PBS, the tissue is incubated overnight at 4°C in the appropriate secondary antibody coupled to either Cy3 or Cy2 (Jackson ImmunoResearch Laboratories, West Grove, PA), or to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen, Carlsbad, CA) diluted 1:500 to 1:2000 in PBS, again on a rocking platform. (Keep dark.)

The tissue is washed again three times for 1 h each in PBS and then carefully placed on a slide, photoreceptor side up. Using a fine paintbrush, the retina is gently flattened and unfolded and then coverslipped with AMM.

The coverslipped slides are left in the dark overnight to harden before oil immersion lenses are used. The edges of the coverslip are sealed with nail polish.

Slides are viewed on a confocal laser scanning microscope (such as the Olympus FluoView 1000) using 40× or 60× oil immersion objectives.