Envisiontm

Immunohistochemical analysis was performed on human tissue microarrays (TMA, RayBiotech, Inc., Norcross, GA, USA). For antigen retrieval, sections were heated in Tris-EDTA buffer (pH = 9) for 10 min and incubated for 1 h at room temperature with mouse anti-HABP2 antibody (1:100) (Novus Biologicals, Littleton, CO, USA). This was followed by 30 min incubation with goat anti-mouse HRP-conjugated IgG (EnVisionTM+, Dako). Slides were developed for 5 min with 3,3′-diaminobenzidene chromogen and counterstained with hematoxylin. Five samples per condition were utilized.

Tissue samples from xenograft tumors (including xenograft subcutaneous tumor model, xenograft metastatic model and PDX model) were fixed in 4% formaldehyde for 24 hours, processed to the paraffin, and sectioned. For immunohistochemistry, 5 μm sections were deparaffinized in xylene and hydrated in alcohol. Endogenous peroxidase activity was then blocked by incubation in 3% hydrogen peroxide-methanol for 10 minutes. For the antigen recovery, the section was heated in a citrate buffer (10 mM Sodium Citrate, pH 6.0) for 15 minutes. After blocking, the primary antibodies against caspase-3(1:1000) (Lot: #9662, Cell Signaling), cleaved caspase-3 (5A1E) (1:400) (Lot: #9664, Cell Signaling) was applied at 4℃ overnight. EnVision+ TM (Dako) was used as the secondary antibody. Antibody binding was visualized by a standard streptavidin immunoperoxidase reaction. These sections were counterstained with hematoxylin.
The score of immunohistochemistry was graded by histochemistry score (H-Score) system. Evaluation was carried out independently by two experienced pathologists. The H-score system was the combination of proportional and intensity of positive tumor cells. H score = ΣPi(i+1). Pi, the ratio of the positive staining cell in all the tumor cells; i, the staining intensity.

Formalin-fixed human lung tissue sections were de-paraffinized in a xylene series and rehydrated through a decreasing ethanol series for immunohistochemistry. The slides were pre-treated by microwave in citrate buffer (100 mM, pH 7.0) for 10 minutes, washed 3x with 1x PBS and 0.1% tween (TBS). Slides were incubated overnight at 4 °C in an anti-Grp78 antibody dilution (1:100) from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). EnVisionTM (DAKO, Bollschweil, Germany) was used for staining and detection, according to the manufacturer’s instructions. A Leica DMI 4000 D microscope was used to acquire images. For double immunofluorescence, tissue sections were incubated with primary antibodies specific to GRP78 (1:100) or SP-C (1:100) (surfactant protein C) (both: Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies used were anti-goat Cy3 labelled to detect GRP78, and anti-rabbit FITC labelled to detect SP-C (Abcam, USA); both secondary antibodies were diluted 1:1000.

Tissue samples from xenograft tumors (including xenograft subcutaneous tumor model, xenograft metastatic model and PDX model) were fixed in 4% formaldehyde for 24 hours, processed to the paraffin, and sectioned. For immunohistochemistry, 5 μm sections were deparaffinized in xylene and hydrated in alcohol. Endogenous peroxidase activity was then blocked by incubation in 3% hydrogen peroxide-methanol for 10 minutes. For the antigen recovery, the section was heated in a citrate buffer (10 mM Sodium Citrate, pH 6.0) for 15 minutes. After blocking, the primary antibodies against caspase-3(1:1000) (Lot: #9662, Cell Signaling), cleaved caspase-3 (5A1E) (1:400) (Lot: #9664, Cell Signaling) was applied at 4℃ overnight. EnVision+ TM (Dako) was used as the secondary antibody. Antibody binding was visualized by a standard streptavidin immunoperoxidase reaction. These sections were counterstained with hematoxylin.
The score of immunohistochemistry was graded by histochemistry score (H-Score) system. Evaluation was carried out independently by two experienced pathologists. The H-score system was the combination of proportional and intensity of positive tumor cells. H score = ΣPi(i+1). Pi, the ratio of the positive staining cell in all the tumor cells; i, the staining intensity.

At the moment of sacrificing, the left kidney was taken for the morphological analysis. Renal tissue was prepared as described previously [8] (link), and stained by hematoxiline eosine (H&E) and periodic acid-Schiff (PAS). Intensity and spread of tubular necrosis, number of intra-luminal cast formations, swelling and vacuolization of cells, loss of luminal membrane or brush borders, tubular dilatation, interstitial oedema and separation of cells from tubular basal membrane were semi-quantitatively evaluated as described previously [8] (link). The level of each manifestation was graded with 1 for low, 2 for moderate, 3 for high, and 0 for the lack of manifestation. The sum of these changes represented the histopathological score.
For investigation of Bax and Bcl-2 expression paraffin, sections were treated by microwave for 20 min at 400 W in citrate buffer (pH 6.0) after deparaffinization and dehydration. After antigen retrieval, samples were incubated with Bax (dilution 1∶250, Millipore, Billerica, MA) and Bcl-2 (dilution 1∶200, Millipore, Billerica, MA) antibodies for 1 hour at room temperature. The EnVisionTM staining method (DAKO) was performed, followed by counterstaining with hemalaun (Merck). Negative controls were performed by omitting the first antibody. The slides were evaluated using the light microscope BX53 (Olympus).

Immunohistochemistry was performed on tissue microarray slides. After deparaffinization in xylene and hydration, the slides were placed in citrate buffer (pH 6.0) and exposed to microwaves for 20 min at 400 W. Peroxidase activity was blocked with 1% BSA (bovine serum albumin). After antigen retrieval, incubation with primary antibodies Slug (1:100, ab27568, Abcam, Boston, MA, USA) and Snail (1:100, PA5-11923, Thermo Fischer Scientific, Waltham, MA, USA) was performed for 1 hour. EnVision^TM (DAKO, Copenhagen, Denmark) was used to visualize the antigen–antibody reaction with 3,3′-diaminobenzine (DAB) and subsequent contrast with hemalaun (Merz, WI, USA). Negative controls were obtained by omitting the primary antibody. Slides were examined using a BX53 light microscope with a DP12CCD camera (Olymus, Hamburg, Germany).
Immunostaining of both markers was independently evaluated by three pathologists (M.Z., G.N., D.D), who were blinded to the patient outcome and pathological information. Valid immunoreactivity was considered as follows: nuclear staining of Slug and Snail, as well as cytoplasmic staining along with nuclear immunopositivity. Tumors were considered to be positive when at least one tumor cylinder in TMA was positive, with more than 50% positive tumor cells, and with at least moderate intensity of the staining. A consensus was achieved for all samples.

The methods of immunohistochemical staining were as described previously [21 (link),23 (link),30 (link)]. Briefly, immunohistochemical staining was performed using 3 μm-thick formalin-fixed paraffin-embedded sequential sections as follows. Samples were first deparaffinized in xylene and dehydrated in graded alcohols. After rinsing in TBS buffer (25 mM Tris–HCl (pH 7.4), 137 mM NaCl, and 2.7 mM KCl), the sections were processed for antigen retrieval in a pepsin solution (Nichirei) at 37°C for 10 min (for EGFR) in a 1 mM ethylenedyamine tetra-acetic acid (EDTA) retrieval solution (pH 9.0) (45211 Nichirei), at 95°C for 40 min (for AMAP1 and GEP100). Endogenous peroxidase was then blocked by incubation in 0.3% H₂O₂-methanol at room temperature for 10 min. After rinsing with TBS, sections were then incubated with primary antibodies against EGFR (1:50), AMAP1 (1:500) or GEP100 (1:100) for 30 min, then with EnVisionTM (Dako, Tokyo) for 30 min, and finally with peroxidase-conjugated streptavidin (Vector Labs, Burlingame, CA) for 50 min. After rinsing in TBS, the coloring reaction was performed with DAB (Dojin, Kumamoto) for 5 min. Each section was counterstained with hematoxylin. These processes were all performed at room temperature.