GeneX and GeneY Expression: Hepatitis C Infected Liver & Control

Fred A. Researcher, Ph.D. Pharmaceutical Company

GeneX and GeneY Summary

Here are slides exposed to emulsion for the GeneX and GeneY in situ hybridization experiment on sections

of three HCV positive human liver samples (1DEC, 1E25, 0243) and normal human liver (6804). Images were from 3 and 4 week exposures to emulsion except for the GAPDH controls that were 2 week exposures.

The positive control probe used to validate the RNA integrity of the four tissues provided was GAPDH,

glyceraldehyde phosphate dehydrogenase, which is a metabolic enzyme expressed in hepatocytes (Matsumura, et al. 2001 Hepatology Research 20:84-96).

In summary, the expression patterns of GeneX mRNAs and GeneY mRNAs overlapped in hepatocytes of

the hepatitis C infected liver samples. GeneY mRNA was not detected in normal human liver. Additional Information

The first set of results was photographed in darkfield illumination at low magnification (2.5x) to demonstrate the overall pattern of hybridization in the sections.

L1 and L2 indicate two independent liver lobules that were traced through the series of sections hybridized with different probes.

HCV+ Liver Sample #1

Slide 02_1DEC_GeneX_AS Slide 03_1DEC_GeneX_S

Slide 04_1DEC_GeneY_AS Slide 05_1DEC_GeneY_S

Slide 02 1DEC GeneX AS was hybridized to the GeneX antisense probe. The ISH signal detected was over the lobules of hepatocytes. Slide 03 1DEC GeneX S was hybridized to the sense control probe to demonstrate the non-specific background hybridization of the probe. The background level of ISH signal for this assay was low. GAPDH results for HCV+ Liver Sample #1 are shown on page 7 (Slide 01 1DEC GAPDH AS).

Slide 04 1DEC GeneY AS was hybridized to the probe for a hepatitis C virus gene product. Slide 05 1DEC GeneY S was hybridized to the sense control probe. It shows the background hybridization signal of this probe.

HCV+ Liver Sample #2

Slide 07_1E25_GeneX_AS Slide 08_1E25_GeneX_S

Slide 09_1E25_GeneY_AS Slide 10_1E25_GeneY_S

Slide 07 1E25 GeneX AS was hybridized to the GeneX antisense probe. The ISH signal detected was over the lobules of hepatocytes. Slide 08 1E25 GeneX S was hybridized to the sense control probe to demonstrate the non-specific background hybridization of the probe. The background level of ISH signal for this assay was low. GAPDH results for HCV+ Liver Sample #3 are shown on page 7 (Slide 06 1E25 GAPDH AS).

Slide 09 1E25 GeneY AS was hybridized to the probe for a hepatitis C virus gene product. Slide 10 1E25 GeneY S was hybridized to the sense control probe. It shows the background hybridization signal of this probe.

HCV+ Liver Sample #3

Slide 12_0243_GeneX_AS Slide 13_0243_GeneX_S

Slide 14_0243_GeneY_AS Slide 15_0243_GeneY_S

Slide 12 0243 GeneX AS was hybridized to the GeneX antisense probe. The ISH signal detected was over the lobules of hepatocytes. Slide 13 0243 GeneX S was hybridized to the sense control probe to demonstrate the non-specific background hybridization of the probe. The background level of ISH signal for this sample was slightly higher than the others. GAPDH results for HCV+ Liver Sample #3 are shown on page 7 (Slide 11 0243 GAPDH AS).

Slide 14 0243 GeneY AS was hybridized to the probe for a hepatitis C virus gene product. Slide 15 0243 GeneY S was hybridized to the sense control probe. It shows the background hybridization signal of this probe.

Normal Liver Sample

Slide 17_6804_GeneX_AS Slide 18_6804_GeneX_S

Slide 19_6804_GeneY_AS Slide 20_6804_GeneY_S

Slide 17 6804 GeneX AS was hybridized to the GeneX antisense probe. The ISH signal detected was over the lobules of hepatocytes. Slide 18 6804 GI-GPx S was hybridized to the sense control probe to demonstrate the non-specific background hybridization of the probe. The background level of ISH signal for this assay was low. GAPDH results for HCV+ Liver Sample #1 are shown on page 7 (Slide 16 6804 GAPDH AS).

Slide 19 6804 GeneY AS was hybridized to the probe for a hepatitis C virus gene product. There was no signal above background for this probe on normal tissue. Slide 20 6804 GeneY S was hybridized to the sense control probe. It shows the background hybridization signal of this probe.

GAPDH Expression

Slide 01_1DEC_GAPDH_AS Slide 06_1E25_GAPDH_AS

Slide 11_0243_GAPDH_AS Slide 16_6804_GAPDH_AS

Slide 01 1DEC GAPDH AS was hybridized to the GAPDH probe in HCV+ Liver Sample #1. Slide 06 1E25 GAPDH AS was hybridized to the GAPDH probe in HCV+ Liver Sample #2. Slide 11 0243 GAPDH AS was hybridized to the GAPDH probe in HCV+ Liver Sample #3. Slide 16 6804 GAPDH AS was hybridized to the GAPDH probe in Normal Liver Sample. The results show the ISH signal was moderate over the hepatocytes for all tissue samples investigated.

HCV+ Liver Sample #1 (10x)

Slide 21_1DEC_GeneX_AS_10x Slide 24_1DEC_GeneY_AS_10x

Slide 22_1DEC_GeneX_AS_DF_10x Slide 25_1DEC_GeneY_AS_DF_10x

The same sections were photographed at 10x magnification to show the localization of expression of GeneX and GeneY in hepatocytes and other cell types in the liver.

Each lobe of the liver is divided into lobules that are roughly hexagonal in shape. At the periphery of each lobule is the portal system, which consists of arteries, veins and lymphatics that supply the hepatocytes in the lobules. There was prominent connective tissue between the lobules of the HCV+ liver samples, but not the normal sample.

Slide 21 1DEC GeneX AS 10x shows a portion of a hepatic lobule (HL) and some adjacent portal vessels (PV). Slide 22 1DEC GeneX AS DF 10x shows the darkfield micrograph of the same section. GeneX expression was detected in the hepatic lobule. The portal vessels were negative. Slide 23 1DEC GeneX S DF 10x shows the background level of ISH signal for this probe (next page).

Slide 24 1DEC GeneY AS 10x shows a portion of a hepatic lobule (HL) and some adjacent portal vessels (PV). Slide 25 1DEC GeneY AS DF 10x shows the darkfield micrograph of the same section. GeneY expression was detected in the hepatic lobule. Cells in the region of the portal vessels were also positive for GeneY, but the expression level was lower than that in the hepatocytes. Slide 26 1DEC GeneY S DF 10x shows the background level of ISH signal for this probe (next page).

HCV+ Liver Sample #1 (10x, continued)

Slide 23_1DEC_GeneX_S_DF_10x Slide 26_1DEC_GeneY_S_DF_10x

HCV+ Liver Sample #2 (10x)

Slide 27_1E25_GeneX_AS_10x Slide 30_1E25_GeneY_AS_10x

Slide 28_1E25_GeneX_AS_DF_10x Slide 31_1E25_GeneY_AS_DF_10x

Slide 27 1E25 GeneX AS 10x shows a portion of two hepatic lobules (HL) and some adjacent portal vessels (PV). Slide 28 1E25 GeneX AS DF 10x shows the darkfield micrograph of the same section. GeneX expression was detected in the hepatic lobules. The portal vessels were negative. Slide 29 1E25 GeneX S DF 10x shows the background level of ISH signal for this probe (next page).

Slide 30 1E25 GeneY AS 10x shows a portion of two hepatic lobules (HL) and some adjacent portal vessels (PV). Slide 31 1E25 GeneY AS DF 10x shows the darkfield micrograph of the same section. GeneY expression was detected in the hepatic lobules. Cells in the region of the portal vessels were also positive for GeneY, but the expression level was lower than that in the hepatocytes. Slide 32 1E25 GeneY S DF 10x shows the background level of ISH signal for this probe (next page).

HCV+ Liver Sample #2 (10x, continued)

Slide 29_1E25_GeneX_S_DF_10x Slide 32_1E25_GeneY_S_DF_10x

HCV+ Liver Sample #3 (10x)

Slide 33_0243_GeneX_AS_10X Slide 36_0243_GeneY_AS_10X

Slide 34_0243_GeneX_AS_DF_10X Slide 37_0243_GeneY_AS_DF_10X

Slide 33 0243 GeneX AS 10x shows a portion of one hepatic lobule (HL) and some adjacent portal vessels (PV). Slide 34 0243 GeneX AS DF 10x shows the darkfield micrograph of the same section. GeneX expression was detected in the hepatic lobules. Slide 35 0243 GeneX S DF 10x shows the background level of ISH signal for this sample that was slightly higher than the others (next page).

Slide 36 0243 GeneY AS 10x shows a portion of one hepatic lobule (HL) and some adjacent portal vessels (PV). Slide 37 0243 GeneY AS DF 10x shows the darkfield micrograph of the same section. GeneY expression was detected in the hepatic lobules. Cells in the region of the portal vessels were also positive for GeneY, but the expression level was lower than that in the hepatocytes. Slide 38 0243 GeneY S DF 10x shows the background level of ISH signal for this probe (next page).

HCV+ Liver Sample #3 (10x, continued)

Slide 35_0243_GeneX_S_DF_10X Slide 38_0243_GeneY_S_DF_10X

Normal Liver Sample (10x, continued)

Slide 39_6804_GeneX_AS_10X Slide 42_6804_GeneY_AS_10X

Slide 40_6804_GeneX_AS_DF_10X Slide 43_6804_GeneY_AS_DF_10X

Slide 39 6804 GeneX AS 10x shows a portion of one hepatic lobule (HL) and an adjacent portal vessel (PV). Slide 40 6804 GeneX AS DF 10x shows the darkfield micrograph of the same section. GeneX expression was detected in the hepatic lobule. Slide 41 6804 GeneX S DF 10x shows the background level of ISH signal for this probe.

Slide 42 6804 GeneY AS 10x shows a portion of one hepatic lobule (HL) and an adjacent portal vessel (PV). Slide 43 6804 GeneY AS DF 10x shows the darkfield micrograph of the same section. GeneY expression was not detected in the hepatic lobule or the portal vessel. Slide 44 6804 GeneY S DF 10x shows the background level of ISH signal for this probe.

Normal Liver Sample (10x, continued)

Slide 41_6804_GeneX_S_DF_10X Slide 44_6804_GeneY_S_DF_10X

Methods, Storage & Viewing

Methodology Tissue Fixation, Embedding and Pretreatment

Tissues were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) overnight, dehydrated and infiltrated with paraffin. 5 to 7 micron serial sections were mounted on gelatinized slides. 1 to 3 sections were mounted/slide, deparaffinized in xylene, rehydrated and post-fixed. The sections were digested with proteinase K, post-fixed, treated with triethanolamine/acetic anhydride, washed and dehydrated.

cRNA probe preparation The cRNA transcripts were synthesized according to manufacturer's conditions (Ambion) and labelled with

35S-UTP (>1000 Ci/mmol; Amersham). cRNA transcripts larger than 200 nucleotides were subjected to alkali hydrolysis to give a mean size of 70 bases for efficient hybridization.

Hybridization and Washing Procedures Sections were hybridized overnight at 52°C in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl pH

7.4, 5 mM EDTA, 10 mM NaH2PO4, 10% dextran sulfate, 1X Denhardt's, 50 µg/ml total yeast RNA, and 50-75,000 cpm/µl 35S-labelled cRNA probe. The tissue was subjected to stringent washing at 65°C in 50% formamide, 2X SSC, 10 mM DTT and washed in PBS before treatment with 20 µg/ml RNAse A at 37°C for 30 minutes. Following washes in 2X SSC and 0.1X SSC for 10 minutes at 37°C, the slides were dehydrated and dipped in Kodak NTB-2 nuclear track emulsion and exposed for one week in light-tight boxes with dessicant at 4°C.

Imaging Photographic development was carried out in Kodak D-19. Slides were counterstained lightly with toluidine

blue and analyzed using both light- and darkfield optics of a Zeiss Axiophot microscope. Sense control cRNA probes (identical to the mRNAs) always gave background levels of hybridization signal. Storage & Rehydration

For any section that is "crystallized", it may be repaired by allowing the coverslip to fall off after soaking in xylene for 24-48 hours. Rehydrate the slide to 70% EtOH. Redehydrate the slide in 80%, 95% and 2x 100% EtOH for 2 minutes each. After three changes of xylene, mount the coverslip with Cytoseal (VWR Scientific) or other comparable mounting medium. Using the same method, coverslips can be removed for histological staining to take brightfield micrographs. Histological stains that require acidic conditions may dissolve silver grains. Overstaining may obscure the silver grains. Any excess mounting medium or residual emulsion on the back of the slide can be removed with a single-edged razor. Dry the recoverslipped slides flat for 24 hours. These slides can be stored indefinitely at room temp. Viewing Original Slides

The results are best viewed with darkfield illumination, but with a 20x or 40x phase-contrast objective, the silver grains can be localized over particular cell groups. The antisense probe (AS) detects the mRNA and the sense control probe (S) shows the background level of silver grains for the experiments. Slides have been digitally captured into Photoshop 6.0 TIFF files and labeled with the tissue and whether the result was with the antisense or sense probe; a, b, c, etc. indicate a different region of the section on the same glass slide. Scans of a one millimeter micrometer, taken with the same objective as the figures, are provided if scale bars are required. The large lines on the micrometer represent 100 microns. All micrographs are 2.5x unless noted.