This expansion microscopic protocol enables the investigator to visualize glomerular proteins with nanoscale resolution on a conventional microscope. The main advantage of expansion microscopy is that it is accessible to a large research community. Begin by making spacers for the gelation chamber.
Cut number one and number 1.5 glass cover slips into five millimeter stripes, using a diamond knife. Place a glass slide into a staining chamber and position the number 1.5 cover slip stripes on the glass slide, so that they form a 2.5 centimeter square. Pipette a droplet of double distilled water into the corners of the square to adhere the glass cover slip stripes to each other and to the glass slide.
Wait for approximately 20 minutes so that the number 1.5 cover slips are stably attached. Pipette a droplet of water on each number 1.5 cover slip stripe. Place four number one glass cover slip stripes onto the number 1.5 cover slips.
For the gelation chamber lid, wrap a cover glass with paraffin film, avoiding any folds or dirt on the film. For anchoring treatment, remove PBS from the immune stained cells and add 250 microliters of anchoring buffer per well directly onto the glass cover slip. Incubate the plate for three hours at room temperature.
Keep the substrate in the dark using a box. After the incubation, remove the anchoring buffer and wash the cover slip once with 1.5 milliliters of PBS per well. To stain actin fibers, thaw an EXM compatible phalloidin solution and incubate it for 45 minutes at room temperature.
Meanwhile, dissolve sodium acrylate in double distilled water using a stirring device and prepare the monomer solution on ice. Remove phalloidin from the cells and wash them with 1.5 milliliters of PBS, twice, at room temperature. Leave 1.5 milliliters of PBS within the well after the last wash.
Use forceps and a cannula to lift the cover glass slip from the six well plate and place it into the gelation chamber. Add APS to the gelling solution and vortex briefly. Pipette 200 microliters of gelling solution on the sample and cautiously close the chamber, avoiding air bubbles within the gel.
Add water to the staining chamber, and incubate the staining chamber for at least one hour at 37 degrees Celsius to polymerize the gel. Take the staining chamber out of the incubator. To open the gelation chamber lid, introduce a razor blade between the lid and the spacer, and remove the lid cautiously.
Remove the spacers with the razor blade and cut off all extra gel. Use a paint brush to detach the edges of the gel. Put the slide with the gel and cover glass into a dish filled with PBS.
Then remove the detached cover glass from the gel by shaking it gently. Put the slide below the gel to attach the gel to the slide. With the gel on the slide, divide the gel into small pieces using the razor blade.
Gently push one piece of gel into a well of a six well plate with a glass bottom and unfold it with a paintbrush. Use the paintbrush to keep the gel moisturized with a small amount of PBS. Using an inverted microscope, take overview images with low numerical aperture for pre-expansion images.
Dilute Proteinase K to four units per milliliter in digestion buffer to make the digestion solution. Add 500 microliters of the digestion solution to each well, and immerse the gel within the solution. Let it digest overnight at room temperature with the lid closed to keep the samples in the dark.
Remove the digestion solution with a pipette and discard it. Add one milliliter of double distilled water and incubate the immersed gel for 10 minutes at room temperature. Remove the water and add one milliliter of fresh, double distilled water.
Continue exchanging water every 10 minutes until a plateau of expansion is reached, causing the gel to become optically clear. Remove the water from the gel and immediately start microscopy. Using an inverted microscope, use an air objective at low magnification to find cells in the pre-expansion state.
Switch to 40 X and 63 X objectives for better resolution. Excite with the wavelength of interest and take the image via the camera. This EXM protocol enables expansion of up to four fold.
To determine the expansion factor it is essential to image cells before and after expansion. Insufficient anchoring and homogenization may lead to distortions and ruptures of cells. Representative examples of ruptured cells are shown here.
This method can be used to investigate the colocalization of F-actin and actin adapter proteins, such as podocin and nephrin. Podocin is depicted in green, actin in blue, and nephrin in red. White areas indicate colocalization.
The fluorescent signal intensity and abundance are key for successful EXM. Efficient anchoring of the fluorescent dyes via ACX is of critical importance for this protocol. In our hands solubilized ACX is good for up to three to four months.
