Clearing and Whole Mount Immunohistochemistry for Smooth Muscle Actin Visualization during Regeneration

Alpha-smooth muscle actin (α-SMA) is a marker of the vascular smooth muscle cells that surround blood vessels, myofibroblasts which respond to injury, and pericytes which are involved in blood vessel maturation and blood flow regulation [1,2]. These cells are important players in tissue healing, regeneration, and regenerative angiogenesis. Whole mount sample preparations have the advantage over paraffin sections of the preservation of three-dimensional context, which is valuable in maintaining the architecture of the vascular bed within regenerating tissues, but the thick nature of these samples necessitates tissue clearing. Immunohistochemical labeling can be coupled with tissue clearing but can present challenges in non-mammalian, non-standard animal models, as the antibodies are usually developed and validated for use in mammalian systems. We sought to develop a whole mount immunohistochemical (IHC) protocol for labelling α-SMA that was compatible with tissue clearing for the amphibian Ambystoma mexicanum as this vertebrate species, unlike mammals, maintains immense regenerative capacity, including the regrowth of entire appendages, throughout its lifespan. Towards this end, we assessed the effect of varying the following parameters: blocking agents (blocks of endogenous peroxidase, serum blocks, non-specific blocks), buffers, nonionic surfactant type and concentration (Tween20 or TritonX) and primary and secondary antibody concentrations. Larval white strain animals (RRID AGSC_101L23; 2-3cm snout-vent length) were purchased from the Ambystoma Genetic Stock Center. All animal procedures were conducted in accordance with an approved IACUC protocol. We chose to use chromogenic IHC methods rather than immunofluorescence, as it allows a more permanent, non-fading preparation in which the surrounding tissue morphology is well preserved. Methods were adapted from Dent et al. [3]. Animals were preserved in 4% paraformaldehyde in phosphate buffered saline (PBS). This was followed by extensive washing in PBS. Bleaching of the specimens was not necessary as the white strain animals possess little brown pigment. We varied the protocol parameters described above; the lowest nonspecific background staining was found using the following conditions. To minimize nonspecific background labeling, we used an endogenous peroxidase block with 10% v/v hydrogen peroxide (1 hour) and a non-specific block with 2% nonfat milk (6% w/v instant nonfat dry milk solids; 30 min.). The samples were also incubated for 90 minutes in blocking buffer consisting of 20% goat serum and 5% nonionic surfactant in PBS. Samples were incubated overnight at room temperature with a 1:10 α-SMA antibody dilution in blocking buffer. After washing, the samples were incubated overnight at room temperature with 1:1000 horse radish peroxidase (HRP) conjugated secondary antibody in blocking buffer. After washing, labeling was visualized using a 3,3’-diaminodbenzidine (DAB) chromogenic substrate kit (Boster Biotech) following the manufacturer’s instructions and a staining time of 10 minutes. Following labeling, samples were optically cleared using a 1:2 solution of benzyl alcohol:benzyl benzoate (BABB). Samples were then stored in BABB. Specimens were imaged with a Bausch & Lomb stereomicroscope and a Zeiss Axiovert 35 inverted microscope both before and after the clearing process. Images were captured using a Celestron ocular camera attachment and Micam software. Images were recorded from both dorsal and ventral regions in the trunk and tail of each animal. ImageJ [4] software was used for image calibration and the addition of scale bars. We found that the clearest labeling was obtained using a high primary antibody concentration along with a multitude of blocking agents (peroxide, serum, and milk protein). The BABB clearing process resulted in some tissue shrinkage; in future iterations it is hoped a more gently graded ascending series of BABB will help minimize this processing artifact. BABB clearing of the tissues improved visualization of the blood vessel network compared to uncleared tissue (Fig. 1 C, D). Further whole mount IHC protocol optimization for non-standard model organisms and more sophisticated higher resolution imaging is expected to be a useful technical tool in characterizing the complex tissue interplay inherent to regeneration [5]. Top row A,B: Low magnification images showing tail tip of secondary only control (A) and α-SMA labeled samples (B). Scale bar: 1 mm. Bottom row, C, D: Higher magnification images of the same sample before (C) and after (D) clearing with BABB. Vascular network is more apparent in the BABB cleared sample. Scale bar=200µm.