Ab4051

Deparaffinized sections (4 μm-thick) from paraffin blocks were separately stained as we described previously37 (link)38 (link) using the following primary antibodies: rabbit polyclonal to Fas Ligand (1:1000, ab15285, Abcam, USA) and rabbit polyclonal to Caspase 3 (1:1000, ab4051, Abcam, USA). Secondary antibody was Envision goat anti-rabbit HRP (DAKO, USA). The evaluation of the immunostaining of these proteins was blinded to the pathologists (Huang J and Feng Z) by an independent observation simultaneous design. The sum of the percentages and intensity scores was used as the final staining score, as described previously22 (link).

Immunolocalization was performed either by a single antibody colourimetric (DAB) immunostaining method, as described previously^{41 (link)}, a single or double antibody tyramide fluorescent immunostaining method, as described previously^{11 (link),42 (link)}, or using an automated Bond immunostaining method, as described previously^{41 (link)}, Exceptions to this protocol are the H₂O₂ concentration washes performed at 3% H₂0₂ TBS. Antibodies used are listed in Table 1. A minimum of five individual sections for each genotype were immunostained in each experiment.Table 1

Immunohistochemistry performed in this study, listing antibody source and method used.

Protein stained for	Method	Primary antibodies
PCNA	DAB	Sigma #P-8825
AKR1B7	Single fluorescence	Santa Cruz Biotechnology #sc-27763
HSD20alpha	Single fluorescence	Aviva Systems Biology #40002
GR	DAB	Cell Signalling #12041
GR/GFP	Double fluorescence	Cell Signalling #12041, Thermofisher scientific #a11122
CASP3	Bond	CASP3: Abcam #ab4051

The cerebral cortex and cerebellum of four animals from each experimental group were dissected out after formalin perfusion. Dehydration, clearing and paraffin embedding were followed according to [25 ]. Sections were stained with haematoxylin/eosin and examined under a light microscope.
For immunehistochemical staining, successive 5 μm thick cerebral cortex paraffin sections were loaded on positively charged slides and incubated overnight at 37 _C, to assess the expression of caspase-3, Bcl-2 according to technique described by Hsu et al. [26 (link)] and GFAP according to [27 (link)]. Primary antibodies of caspase-3 (ab4051, abcam, Inc), Bcl2 (ab196495, abcam, Inc) and GFAP (Ab-1,Clone GA-5, Lab Vision Corporation, Medico Co., Egypt) were applied for immuno-localisation according to the manufacturer's protocol. For immunostaining of synaptophysin, deparaffinized sections of cerebellar cortex were incubated with rabbit polyclonal primary antibody; Synaptophysin antibody(PA5-27,286, Thermo Scientific, USA). Binding of primary antibody was demonstrated using Thermo Scientific Super Picture™ Polymer Detection Kit.

After 48 h of culturing human mammary cell lines in CM, cell line medium, or DMEM, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro toxicology assay or a lactate dehydrogenase (LDH) assay was carried out, as previously described^{11 (link)}, according to manufacturer’s instructions (Sigma). MTT and LDH absorbances were measured at 570 nm and 490 nm, respectively, on a Multiskan EX plate reader (Thermo Scientific, Vantaa, Finland) and background measurements of 690 nm were subtracted for both. Optical densities of wells treated with EqMDEC CM were compared to those treated with either DMEM or control (self-CM) in order to determine cell viability or relative LDH release. Values were expressed relative to wells treated with DMEM and to lysed wells for MTT and LDH release, respectively. For active caspase 3 immunostaining, 4% paraformaldehyde-fixed cells were washed with PBS and treated with 0.5% Triton X-100 for 10 min. Following a 30 min incubation in blocking solution (1% goat serum and 1% BSA in PBS) at RT, fixed cultures were probed with an anti-active caspase 3 primary antibody diluted 1:100 in PBS (ab4051, Abcam, Cambridge, MA) for 1 h, followed by incubation with HRP-conjugated goat anti-rabbit diluted 1:100 in PBS (Jackson ImmunoResearch, West Grove, PA) for 1 h.

Sections from liver and lung in the above mouse models were fixed, embedded, sectioned, and stained with H&E according to the standard protocol. Ki-67 staining was performed according to the manufacturer’s protocol (mouse monoclonal antibody, 1 : 500, Sc-25280, Santa Cruz Biotechnology, Dallas, TX, USA). For apoptosis detection, Caspase-3staining was performed according to the manufacturer’s instruction (Rabbit polyclonal antibody, 1:1000, ab4051, Abcam, Cambridge, MA, USA). Oil-red-O (ORO)-staining was performed according to the standard protocol.^{24 (link), 33 (link)}