Oregon Green 488 BAPTA-1 can detect large changes of intracellular calcium concentrations and it is therefore useful for intracellular loading by microinjection. Today, we did exactly this.
Surgical procedure
An adult wild type mice was injected using a dose of pentobarbital sodium (1% pentobarbital sodium). Local anesthetic was also used around the scalp using lidocaine. The scalp of the mouse was exposed and its head fixed to the steotaxic apparatus making sure that the external auditory canal of thought were not damaged. There could be a number of challenges one would have to be aware of when fixing the head of a mouse on the stereotaxic apparatus. One would have to make sure that the animal is fully anaestatise so it will not wake up. It is also good to make sure that the brain is flat.
Specific site of the brain is located by first finding the bregma. It is an important landmark used in neuroscience because it is the 0 starting point of any experiment in order to carry out local microinjection loading at a desired location, in our case the visual cortex. We aimed to go at V1 AP:- 3.2 mm; L: 2.6 mm. To do this, the pipette was placed and position at the bregma site and from there, the pippete was navigated to the visual cortex (V5). A small hole was of a 1 mm in diameter was drilled. This is to be as small as possible so that…An 80 nL of dye was slowly inserted using the pippete to the specific depth of the cortex at Layer V of aroun 500 um. The dye was injected manually but in a more in controlled experiments usually one would use a pump at a rate of around 50 um/s. Normally one would have to wait around 30 mins for ensuring the dye’s homogenous dispersion within the local site but due to time constraint in this exp., we waited around 10 mins. The hole was filled with a wax. The scalp was glued. The mouse was perfused through the heart with saline sol (40 cm3), followed by PFA to fix the brain (40 cm3). Perfusing was done as quickly as possible and ideally done before the mouse’s heart stops beating entirely.
The brain slices were obtained using the vibratome. It cuts thin slices of 80 um. Each slices were collected in order and soaked in PBS containing Hoescht 332 for nucleus staining. Knowing the the injection was at the depth of around 500 um, some of the slices were selectively fixed into glass slides and imaging and finding the injection site by epifluorescent microscope.
© 2014 So you think you can grow crystals in a beaker.
* Experiments were carried out under the institutional guidelines of the Third Military Medical University, Chongqing as part of the Sino-German Summer Course “two-photon functional imaging of the living brain” as part of a demo session.
Roberts Lab 