Biological systems are capable of forming a complex neural network of forwarding feeding, feedback, and horizontal circuits ; the most sensitive biological systems are the nerve base of spatial codes . For decades, research on the neurobiology of neural tissue imaging focused mainly on anatomical structures of the nerves by mechanical slicing techniques, which laid the foundations for understanding the neural maps of two dimensional (2D) spatial displays . However, texture transparency and light scattering greatly limit the depth of the tissue that can be drawn up optically .
The three dimensional (3D) image has enabled studying cellular and extracellular structures, such as the structure of vasculature, or neuronal networks in the brain [3, 4]. Various protocols have been developed to clean the entire tissue and 3D imaging . Benzyl alcohol and benzyl benzoate (BABB) were among the first materials that made 2 cm thick fixed tissue transparent for deep microscopic imaging and were used in conjunction with conventional immunohistochemical techniques compared to tissues of less than 50 μm [5, 6]. Besides, numerous advances have been made for high resolution and large-scale imaging of cleared tissue, including Scale , dibenzyl ether (DBE) , 3D imaging of solvent cleared organs (3DISCO) , See Deep Brain (seeDB) , Clear T , Clear Unobstructed Brain/Body Imaging Cocktails (CUBIC) , System-Wide control of Interaction Time and kinetics of Chemical (SWITCH)  and ultimate DISCO (uDISCO) . The aforementioned techniques require a very clear texture and transparent tissue to detect .
Considering the limitations of the above-mentioned techniques, including the isolation of fluorescence from samples, incomplete cleansing samples, and the lack of feasibility for antibody labeling, several other techniques have been developed . However, these protocols were still limited by the isolation of fluorescence from samples, the inadequate purification of samples, and the inability to allow antibody labeling . Efforts to address these issues and to modify tissue processing conditions have provided the primary inputs for optical clearing techniques . The cell membranes as the main source of light scattering in tissues and lipid removal, is a potential way to increase tissue transparency .
Several techniques have been developed to eliminate the transparency of lipid for 3D imaging, including the use of acrylamide protocols such as CLARITY , passive CLARITY , passive clarity technique (PACT) , and Perfusion-Assisted Agent Release in Situ (PARS) . These methods use embedding hydrogel such as CLARITY and PACT . These hydrogel-based techniques also require toxic chemicals, labor work, and specific lab equipment. Not only they are expensive, but also they change the volume of the texture even after using the refractive index matching solutions (RIMs) . The cutting edge technique CLARITY developed by Chung and Deisseroth  has created a new tissue processing platform to illuminate a 3D cell conjugation and make-up in general . This clearing technique, the most commonly used method for the study of healthy tissue image and can be applied to explore structural molecular and intact tissue infrastructure, is largely constrained due to the use of tissue-specific reagents or special application . Three transparent roles have been created through these techniques: the stabilization of tissue structures using embedding hydrogel , the use of large-scale compatible clearing reagents , and enhanced tissue imaging [2, 18]. Whole-tissue clearing takes days to weeks to disrupt the fluorescent signaling of the labeled chemical, and ultimately cannot prevent the separation of fluorescent protein signals for a long time. These limitations are more problematic in developing countries laboratories with limited equipment, so, a simple method is needed for laboratories in developing countries .
The other techniques have been developed without applying acrylamide methods including Free of Acrylamide Sodium Fast Free-of-Acrylamide Clearing Tissue (FACT)  and Dodecyl Sulphate (SDS)-based Tissue Clearing (FASTClear) . The FACT requires less labor, and toxic and harmful chemicals to the environment than the acrylamide-based ones . Another limitation in developing countries is the lack of advanced microscopes, i.e. confocal, two-photon excitation, and light-sheet microscopes . To date, all protocols for 3D imaging of tissues have been used by advanced microscopes. The use of the FACT approach with a conventional epifluorescence microscope is another goal . Therefore, there are two procedures described in this paper that the goal of the first procedure is to speed up the structural analysis of whole tissue clearing and to apply to the fluorescent imaging of mouse brain tissue. The aim of the second procedure is to evaluate the FACT protocol for clearing the entire mouse tissue and 3D imaging of brain cortical vasculature using the FACT method by a simple epifluorescence microscope in imaging laboratories [5, 21, 22]. FACT has also been used for monkey brain imaging using a Zeiss LSM 880 scanning confocal microscope .
Clarifying large tissue volumes
The FACT was firstly obtained by perfusion with phosphate-buffered saline (PBS) and then from 4% paraformaldehyde (PFA) (w/v) in 1 M PBS as a fixative solution. After collecting a 1 mm coronal section of mouse brain sample, brain fragments were supposed to be fixed in a fixative solution at 4°C for 3 days. SDS, which is a highly effective surfactant, was used to remove fatty spots (Figure 1A) . The samples were observed daily until obtaining visual confirmation of full transparency of the tissue by viewing black grid lines on a white sheet of paper through the tissue itself (Figures 1B-D) . Other tissues of mice and rats have been shown in Figures 2 and 3, respectively.
After clearing the fragments of the brain with the FACT protocol, for staining the cleared tissue with the antibodies, SDS was removed by washing in PBS with Triton X 100 (PBST) for 12 h. The PBST solution was replaced every 6 h. Glycine, Triton X 100, donkey serum, and dimethyl sulfoxide (DMSO) dissolved in PBS was injected into the tissues and blocked the permeability at 37°C. The tissues were washed twice in PBST for 1 hour at 37°C. Brain slices were incubated with primary antibodies, Tween-20 (as a nonionic surfactant), DMSO, donkey serum and sodium azide in PBS for at least 2 days at 37°C. The specimens were washed three times in PBST for 1 hour and incubated at 37°C. Then, sections with secondary antibodies were incubated in Tween-20, DMSO, serum, and sodium azide in PBS for at least 2 days . All steps were performed by shaking at 37°C. The specimens were washed three times in PBST for 1 hour and then incubated at 37°C or transferred to a refrigerator (4°C), and thereafter for 7 days in aluminum foil-coated tubes contained PBST and sodium azide .
Imaging large clarified tissue volumes
The next major challenge is the development of high-resolution optical techniques and high-resolution in deep textures . First, frozen coronal brain sections with 30 μm of thickness were prepared on a cryostat [19, 24]. And for the first time, the clearing of the rat tissues with the FACT protocol was shown for effective tissue clearing and 3D imaging of the brain cortex vasculatures  .
The FACT method has undergone some changes, including imaging changes to adapt this method to non-equipping laboratories . The images were taken with a Nikon A1R + upright confocal microscope . Though the main part of the whole-tissue clearing is optical sectioning for 3D imaging that can be achieved optimally with the expensive confocal microscope, the accessibility of this type of microscope is a great challenge for matching the whole tissue imaging in a typical laboratory with limited resources. Therefore, in the FACT method, we used an epifluorescence microscope with a motorized stage for auto-fluorescence vessel imaging in the z plane . However, this approach has limitations, including the lower depth of the fluorescent light image in an epifluorescence microscope compared to a laser in a confocal microscope. This can be solved by cutting 1 to 2 mm of cleared tissue for imaging. In addition, the lower epifluorescence power for collecting high signals compared with the laser-enhanced fluorophores in the confocal imaging of the tissue for a maximum depth of 200 to 300 µm .
Overview of the procedure
We here describe the FACT protocol (Figure 4). Before starting the process, reagents are prepared, and tissues are collected (Step 1 in method#1 and Steps 1-2 in method#2). The tissue clarification process includes fixation of the perfused brain (Steps 2-3 in method#1 and Steps 3-4 in method#2), clearing of the tissue (Steps 4-5 in method#1 and Steps 5-8 in method#2), and RIM (Step 12 in method#1 and Steps 9 in method#2). If required, tissues are going under antibody staining procedure (Steps 6-11 in method#1 and Steps 10-17 in method#2) (Figure 5) in advance of RIM. The cleared tissues undergo confocal microscopy and imaging (Steps 13-16 in method#1 and Steps 18-22 in method#2) (Figures 1E, F), followed by 3D reconstruction and image analysis (Steps 17-23) (Figures 1G, H); we describe the application of FACT protocol, which is suitable for lipid removal in different kinds of cleared tissues.
The lipid removal is crucial for transparency and efficient antibody staining throughout the whole brains. The lipid removal normally is a harmful process and causes loss of biological molecules such as proteins, but now with the FACT technique, chemical bonds of membrane and intracellular cytoplasmic proteins with the cytoskeleton and the extracellular matrix assist the creation of a massive 3D matrix and structural support to fortify the tissue during processing (Figure 6) [19, 25].
The hydrogel-based techniques as mentioned earlier require toxic chemicals, labor work, and specific lab equipment. Not only are they expensive, but also they change the volume of the texture even after using the RIMs . The CLARITY developed is the most commonly used method for studying whole tissue image and can be applied for exploring structural molecular and intact tissue infrastructure [2, 17]. Another limitation is the timing in which the whole tissue clearing takes days to weeks to disrupt the fluorescent signaling of the labeled chemical and ultimately cannot prevent the separation of fluorescent protein signals for a long time . On the other hand, The FACT requires less labor, and toxic and harmful chemicals to the environment than the acrylamide-based ones . Additional to that limitation, in developing countries, there is a lack of advanced microscopes, i.e. confocal, two-photon excitation, and light-sheet microscopes . To date, all protocols for 3D imaging of tissues have been used by advanced microscopes. The use of the FACT approach with conventional epifluorescence microscopy is recommended instead of methods using advanced microscopes .
As a final comment for future work, we think that the removal of polyacrylamide hydrogels increases the rate, amount, and depth of antibody penetration and provides a clear image compared to endogenous fluorescent labeling. Maintaining the primary or secondary antibodies in the hydrogel networks or crosslinking of these antibodies with the hydrogel creates unexpected non-specific staining and increases the background during imaging, which significantly reduces the image quality.
There are two protocols for FACT. One with confocal microscopy (Protocol #1) and another one with epifluorescence microscopy (Protocol #2) that can be used depending on the laboratory equipment.