Histomount

The tissue was processed with the classical chrome-osmium rapid Golgi staining method (11 (link),33 (link)-36 (link)). It was placed in 4% paraformaldehyde for 12-18 h and afterwards immersed in rapid Golgi solution consisting of 0.3% osmium tetroxide and 3% potassium dichromate for seven days. The solution was replaced with 1% silver nitrate, in which the tissue was immersed for two days. These steps were performed in a darkened room. The tissue blocks were then dehydrated in an ethanol cascade (70%, 96%, absolute ethanol) and put in alcohol-ether (1:1). The tissue segments were rapidly embedded in celloidin and serially cut on a microtome into coronal slides at a thickness of 200 μm. They were briefly dehydrated (50%, 70%, 96%, ethanol, butanol-ethanol) and placed into Histoclear and mounted with Histomount (National Diagnostics, Atlanta, GA, USA).
The success of Golgi impregnation was determined in accordance with relevant literature (12 (link),34 (link),37 (link),38 (link)). No staining artifacts due to postmortem delay were detected.

Milligan’s trichrome technique was employed for the histological staining of Bouin’s fixed sections with modifications in the timing of staining of acid fuchsin (3 min) and fast green (8 min) [43 (link)]. All sections were de-waxed, rehydrated, stained and mounted with coverslip using Histomount (National Diagnostics) before imaging. Each stage.

Following dissection at the appropriate stage of development, embryos were washed in PBS, and then fixed by immersion in 4% paraformaldehyde in PBS at 4°C for 1 to 3 nights dependent on their age. Embryos were subsequently dehydrated in ethanol and processed for wax sectioning. Sections were cut at 8 µm using a rotary microtome (Leica). For haemotoxylin and eosin staining, slides were de-waxed with two 10 minute washes of Histoclear and were hydrated to water through an ethanol/H₂0 gradient (100%, 90% 70% and 50%). Slides were placed in Ehrlich haematoxylin (RA Lamb) for 10 minutes then were transferred into a trough of running tap water. The slides were left until the sections changed colour from purple to blue. The sections were differentiated by dipping in acid alcohol (1% HCl in 70% ethanol) for 10–30 seconds, and then were placed back into tap water until the blue colour was restored. When an acceptable intensity of haemotoxylin stain was achieved, the slides were transferred into 1% aqueous eosin for 5 minutes, rinsed in tap water, then dehydrated through the same ethanol gradient, before washing twice in Histoclear and mounting in Histomount (National Diagnostics).

Whole embryos, larvae, and dissected adult tail tissues were fixed for 4–5 days in 4% paraformaldehyde (PFA) in PBS, subsequently dehydrated and embedded in paraffin wax (Sigma-Aldrich) using a Shandon Citadel Tissue Processor 2000 (Thermo Scientific), and sectioned to 5μm thickness using a rotary microtome (Leica Biosystems RM2125 RTS). Sections were stained with Haematoxylin and Eosin (H&E) using a Shandon Varistain 24-4 automatic slide stainer (Thermo Scientific), and embedded with Histomount (National Diagnostics). Images of histological sections were captured using a Zeiss Axioskop 40 microscope equipped with an Olympus color camera DP70.

Bovine nasal mucosa were collected for histology examination as described above with a 1 cm square dissected out and snap frozen in liquid nitrogen. Cryosections of 8 μm were fixed with acetone, stained with Vector® Red Alkaline Phosphatase Substrate Kit (Vector Labs, Peterborough, UK), in the presence or absence of levamisole inhibitor (Sigma-Chem Co, Poole, UK) counterstained with Gills Haematoxylin (Sigma-Chem Co, Poole, UK) and mounted with Histomount (National Diagnostics, New Jersey, USA). Images were taken with Olympus BX51 and attached DP71 camera (Olympus, Japan), using Cell^D software (Olympus, Japan).

Immunohistochemical studies were performed on paraffin-embedded tissue sections. Deparaffinization (immunohistochemistry protocol for paraffin-embedded tissue sections, Cell Signaling Technology, MA, USA) was initially carried out, followed by the blocking of nonspecific antigen binding sites by using a universal blocking buffer for one hour at room temperature. Tissue sections were incubated with primary antibodies Rabbit polyclonal RAB25 (Abcam) and Mouse monoclonal Snail (Cell Signaling Technology, MA, USA) for one hour at room temperature, followed by incubation with the respective HRP-tagged secondary antibody for one hour at room temperature. The sections were washed two times for five minutes each with PBST. Immunostaining was detected by using the diaminobenzidine (DAB substrate) method (Cell Signaling Technology, MA, USA) according to the manufacturer's protocol. The peroxidase activity was detected in a DAB working solution containing DAB chromogen concentrate and DAB diluent. The tissue sections were counterstained with Mayer's hematoxylin. The slides were washed with double-distilled water and PBS and mounted with coverslips by using Histomount (National Diagnostics, GA, USA) upon complete drying. The samples were assessed for positive and negative staining by imaging under bright-light microscopy.