Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

Abstract

A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit. Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at -20 degrees C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0 +/- 0.3% only. The loss from dried brain homogenate was 9.5 +/- 1.1%. Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier.

Figure 1

TLC of gangliosides extracted under various conditions. (A) Effect of temperature on dry acetone extractability of GM1 ganglioside. (1) direct extraction with C-M-W; (2) extract with dry acetone at −20°C; (3): C-M-W extract after previous extraction with dry acetone at −20°C; (4): acetone extract at room temperature; (5): C-M-W extract after previous extraction with dry acetone at room temperature. Note: GM1 ganglioside detected with ChTB (GD1b ganglioside band was also detected with much lower sensitivity). GM1 content in all acetone extracts was below the detection level of very sensitive cholera toxin staining. (B) Comparison of GM1 extraction with dry acetone and with aqueous acetone. (6) Direct extraction with C-M-W; (7) extract with dry cold acetone at −20°C. (8): C-M-W extract after previous extraction with dry acetone at −20°C; (9) extracts with mixture acetone-water 9:1 shows significant loss of GM1; (10) C-M-W extracts after previous extraction with acetone-water mixture.

Figure 2

Histochemical detection of GM1 ganglioside in liver cryostat sections after various fixation conditions. Fixation with (A) dry cold acetone for 15 min at −20°C and with (B) 4% formaldehyde for 5 minutes at room temperature gave comparable results. Rat liver cryostat sections stained with cholera toxin B-subunit. Bar 200 µm.

Figure 3

Effect of acetone temperature on histochemical detection of GM1 ganglioside in liver cryostat sections. Fixation with (A) 4% formaldehyde for 5 minutes at room temperature (F); (B) cold dry acetone for 2 minutes and (C) 15 min, respectively; (D) dry acetone at room temperature for 2 min and (E) 15 min, respectively. No apparent differences between GM1 staining intensity or in pattern. Rat liver cryostat sections stained with cholera toxin B-subunit. Bar 100 µm.

Figure 4

Effect of water content in acetone on histochemical detection of GM1 ganglioside in liver cryostat sections. (A) formaldehyde fixation at room temperature for 5 min, (B) dry cold acetone for 15 minutes, (C) cold acetone-water mixture 9:1 for 15 min. Diffusion of the GM1 reaction product after fixation with an acetone-water mixture (C). Rat liver cryostat sections stained with cholera toxin B-subunit. Bar 50 µm.

Figure 5

Compared effect of different fixatives on histochemical detection of GM1 ganglioside in rat liver and brain. Cryostat sections stained with cholera toxin B-subunit. (A, B) Brain cortical grey matter; low power field microphotographs. (A) Diffuse positivity in the neuropil after formaldehyde fixation and (B) focally accented staining after anhydrous acetone + formaldehyde (A+F) combination. Bars 50 µm. (C, D) Brain cortical grey matter; high power field microphotographs. (C) Diffuse staining in the neuropil after formaldehyde fixation. Note also a migration of unstained myelin lipids forming myelin figures (A,C); some of them are marked by arrows. (D) Granular staining after acetone fixation (A+F) suggesting localization of a reactive product to the cell membranes (for a detail see insert in D). Bars 25 µm. (E, F) High power field pictures of the liver sections fixed with (E) formaldehyde or (F) acetone + formaldehyde (A+F). Finely granular staining on the cell membranes of hepatocytes is uninfluenced by the type of fixation. Bars 25 µm. Cell nuclei were counterstained with hematoxylin. (G,H,I) survey of brain cortex; pre-treatment of cryostat sections with (G) formaldehyde, (H) MCD + formaldehyde and (I) anhydrous acetone + formaldehyde. Slight gradual (G →I) decrease in overall staining intensity and increase in sharpness and condensation of the staining pattern. Bars 100 µm.