To begin, embed the transgenic larva in low-melting agarose in a glass-bottom microwell dish. Place the dish onto the microscopic stage at the inverted microscope. Position and focus the desired region for ablation in the center of the field of view.
Choose the Z-stack settings to image the entire breadth of the opercle. Image the opercle area of interest before ablation. Then, draw a circular region with a diameter of 25 micrometers in a selected 2D plane.
Focus on the opercle using the region of interest, or ROI tool. To perform the ablation, apply ablation power measured without objective to 450 milliwatts for a duration of 10 seconds. Expose the selected area to the two-photon laser.
After ablation, confirm the ablation structurally by imaging the same Z-stack with the same settings, as used prior to ablation. Using forceps or a dissection needle, remove the larvae carefully from the low-melting agarose. First, remove a layer of agarose covering the larvae.
Second, remove the agarose in direct contact with the larvae. Place the larvae back in a dish with pure E3 media. At four or six hours post-lesion, re-image the opercle area using agarose-embedded larvae at the same settings on the same microscope.
After processing the images in Fiji, quantify cells with a Nuclear label, or by measuring the area, in case cells are overlapping. To quantify the area, create a mask encompassing the macrophages. Use the Image, Adjust, Threshold Tool, Create a ROI, and analyze the particles.
Then, measure the area. Generate graphs plotting the measurement results using suitable software. A significant accumulation of macrophages in the ablated opercle region was detected six hours post-ablation in six days'post-fertilization larvae.
In larvae four days post-fertilization, the number of macrophages increased at four hours post-lesion. Macrophage number and area increased at four hours post-lesion compared to the un-ablated state.
