GI Adherent Mucous Gel Barrier 57
57
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
5
The Gastrointestinal Adherent Mucous Gel Barrier
Adrian Allen and Jeffrey P. Pearson
1. Introduction
Three phases of production of mucin can be identified in the gastrointestinal (GI)
tract on the basis of their location in vivo: the stored, presecreted, intracellular mucin;
the gel phase adherent to the epithelial surfaces; and the viscous, mobile mucin, which
is largely in soluble form and mixes with the luminal contents. The layer of adherent
mucous gel that lines the epithelial surfaces throughout the gut from the stomach to
the colon marks the interface between the mucosal epithelium and the fluid luminal
environment, which is teeming with nutrients, bacteria, destructive hydrolases, for-
eign compounds and so on. The adherent mucous gel thus provides a protective barrier
and a stable unstirred layer with its own microenvironment, between the mucosal sur-
face and the lumen (1,2). In the mouth, where salivary mucins are secreted, and the
esophagus, there is no discernible adherent mucous gel layer.
A practical definition of the adherent mucous gel layer is that secretion of mucus
which remains attached to the mucosal surface after washing away the luminal con-
tents. This mucous gel layer is readily visible on unfixed, transverse sections of mu-
cosa as a translucent layer of variable thickness (5–200 µm), between the dense
mucosal surface and the bathing solution (3). Mucous gel scraped from the mucosal
surface has the rheological characteristics of a true viscoelastic gel, although it has the
ability to slowly flow (30–120 min) and reform when sectioned (4,5). The concentra-
tion of mucin in such gels scraped from the mucosal surface is high, e.g., ranging from
50 mg/mL in gastric mucus to 20 mg/mL in colonic mucus (6). The extent of the mu-
cus adherent to the mucosal surface in vivo is determined by a balance between the
rate of secretion by the underlying epithelium and the rate of erosion by mechanical

shear associated with the digestive processes and digestion by hydrolases, particularly
proteases (1,2). For full studies of mucous secretion in vivo, it is essential to quantitate
both the adherent mucous gel and mucin in the luminal solution since the latter can
arise by degradation of the former as well as by secretion, and changes in these two
phases do not necessarily parallel each other. Despite being the primary mucous bar-
58 Allen and Pearson
rier and the major secretion of mucin of the gut, the adherent mucous gel is frequently
ignored because of the practical difficulties in observing and quantitating it.
Quantitative measurement of the amount of mucin in the adherent mucous gel
secretion in vivo can only be approximate. Although the mucous gel can be separated
from the mobile mucus by washing the mucosal surfaces, separation of the gel layer
from the considerable intracellular mucin stores cannot be done satisfactorily. The
mucous gel can be removed from the surface by gentle scraping, but inevitably either
not all the adherent gel is removed or the scrapings contain substantial numbers of
epithelial cells with stored intracellular mucin (7). Researchers have attempted to esti-
mate the amount of the adherent mucous gel by utilizing the binding by the dye alcian
blue (AB) to the negatively charged mucin macromolecules (8). However, the amount
of negative charge on mucins varies considerably according to the location in the gut,
secretory status, and disease. Also other macromolecules present in the mucus, e.g.,
negatively charged nucleic acids and cell surface components, will bind the dye, and it
has yet to be shown that the dye penetrates the mucous layer uniformly. Results from
this method are therefore difficult to interpret and this method is not considered fur-
ther here.
The best approach to quantitating changes in the adherent mucous gel layer is by
measuring its thickness. These measurements have the important advantage that con-
tinuity and thickness of the layer can be directly related to functional efficacy (1,2).
Routine histological methods for fixing and staining of mucosal sections, although
readily demonstrating periodic acid-Schiff (PAS)/AB positive intracellular mucin
stores, result in little or no adherent mucous gel being visible at the mucosal surfaces.
This is because the prolonged use of fixatives and organic solvents together with con-

ventional paraffin embedding causes denaturation, dehydration, and loss of the sur-
face mucous gel layer (9–11). Similarly, standard preparation procedures for electron
microscopy distort the mucous layer to give condensed strands or fenestrated patches,
although a continuous mucous layer can be observed by scanning electron microscopy
if it is first stabilized by antibodies (12). The paucity or absence of a mucous layer on
biological sections has led to controversy in the past as to whether an adherent mucous
layer existed over the gastric mucosal surface (13,14). However, a variety of different
methods (discussed subsequently) have now demonstrated unequivocally that a thick
(100–200 µm), continuous, adherent mucous layer exists over the mammalian gastric
mucosal surface. A protective mucoid cap can be seen at the surface of damaged
mucosa undergoing re-epithelialization, following preparation of sections by standard
histological procedures (1,13,15). This mucoid cap has been confused with the adher-
ent mucus that covers the normal undamaged mucosa; however, it is a quite different
structure consisting primarily of a fibrin gel and necrotic cells with some mucin stain-
ing (15).
Original methods for measuring adherent mucous thickness in situ were based on
(1) differences in refractive index between the mucous layer and the mucosal surface,
measured by a slit lamp and pachymeter (16); (2) the dimensions of the surface pH
gradient measured using microelectrodes (17,18); and (3) observation of transverse
sections of unfixed mucosa (3). The first two of these methods also measure the
GI Adherent Mucous Gel Barrier 59
unstirred aqueous layer beyond the adherent mucous gel surface, the extent of which
depends on the hydration of exposed mucosa and the degree of stirring of the solution
above the mucosa, respectively. Consequently, the dimensions of mucous thickness
measured by the slit lamp and pachymeter or pH gradient methods can be substantially
greater than the actual values for the gel layer itself (19). Since the mid-1980s, obser-
vation of the translucent mucous layer on thick (1.6 mm) sections of unfixed mucosa
(detailed subsequently) has been a simple method for measuring mucous thickness,
although histological methodology has now reached the stage where preservation of
full adherent mucous thickness can be maintained during processing (10).

An elegant method for observing mucus and measuring its thickness has been devel-
oped using in vivo microscopy in the anesthetized rat animal model (20–22). The GI
mucosa is exteriorized, with its blood and nerve supply intact, and the mucosa is placed
over an illuminated lucite cone. A lucite chamber is fitted over the mucosa to expose
approx 0.8 cm
2
of the surface and is filled with physiological saline. The mucous layer
covering the mucosa can be observed microscopically from above, particularly if the
luminal surface of the mucus is enhanced visually by the addition of carbon particles
to the bathing solution. The thickness of the mucous layer is best measured by record-
ing the distance travelled by a glass microprobe moved by a micromanipulator at a
fixed angle through the mucous layer. A continuous mucous coat of mean thickness
200–300 µm is seen in rat stomach and duodenum (21,22), and a mean thickness of
800 µm is seen in the colon (23). Strong suction will remove about one-third of the
mucous layer in the stomach and nine-tenths of that in the colon to leave a firm adher-
ent gel, whereas in the duodenum all the mucus can be sucked off. It is not yet clear
whether the mucus that can be sucked off in vivo is a continuum of the gel at a lower
concentration or a separate secretion. This method, which for experimental practicali-
ties can be applied only to the rat (and possibly other experimental animal models), is
particularly useful for validating other in vitro methods for measuring mucous thickness,
for studying mucous secretion in vivo, and for studying the secretion of acid from the
gastric glands through the covering mucous layer (20). Full details of this method, which
requires careful physiological experimentation to ensure full maintenance of in vivo
functions during the experimental period, can be found in refs. 20–22.
Various groups have adapted histological procedures to observe adherent mucus on
fixed mucosal sections. Modifications have included the use of cryostat sections (24)
and fixation in Carnoy’s solution (11,25). However, the adherent mucous layer after
such procedures is very thin (typically about 25%) compared with that seen on unfixed
mucosal sections and even less than that observed in vivo. A particularly interesting
method developed by Ota and Katsuyama (11,25) shows alternating layers within both

human gastric and colonic adherent mucous layers following dual staining with galac-
tose-oxidase-cold thionine Schiff and paradoxical concanavalin A. However, this
method, which suggests some form of substructure within the mucous gel layer, uses
Carnoy’s fixative, as well as clearing in xylene with paraffin embedding, and results in
considerable shrinkage of the mucous gel to give a thickness less than one-fifth that
seen on unfixed sections. A recent method (detailed subsequently), using cryostat sec-
tions, does give thickness values in the rat comparable with those measured for the
60 Allen and Pearson
firm adherent mucous gel remaining after suction in vivo (10). In this method, cryostat
sections receive no prefixation or extensive dehydration steps characteristically used
in conventional staining techniques. Instead, the tissue is given a brief ethanol pre-
treatment followed by a prolonged staining with PAS/AB and a gentle postfixation in
paraformaldehyde vapor, and finally is mounted in a water-soluble gelatin gel. Values
for mucous thickness over the rat gastric mucosa by this method give a mean of
147 µm, which approximates that of the adherent gel remaining after suction in vivo.
It would appear, therefore, that this method preserves intact the thickness of the stable
adherent mucous gel layer. By contrast, mucous thickness by this method is twice that
seen using conventional PAS/AB staining techniques on cryostat sections of mucosa
and 50% greater than that for unfixed rat gastric mucosal sections.
2. Materials
2.1. Mucous Thickness on Unfixed Sections
1. NaCl solution: 150 mmol/L.
2. Two parallel, sharp razor blades separated by spacers 1.6 mm apart such that one side of
each of the two razor blades is left free for sectioning and held together by bolts tightened
by hand.
3. Millipore filter paper to act as a backing for the mucosa during sectioning and subsequent
manipulation of the section.
2.2. Histological Staining and Fixation of Cryostat Sections of Mucosa
1. NaCl solution: 150 mmol/L.
2. Acetic acid solution: 3% (v/v).

3. Alcian Blue 8 GX (Sigma, Poole, Dorset, UK) 1% by weight in 3% acetic acid (pH 2.5).
4. Periodic acid (aqueous): 1%.
5. Schiff’s reagent (fuchsin-sulfite reagent) readily obtained, made up in solution (Sigma).
6. Paraformaldehyde crystals placed in a desiccator for final postfixing of sections at 37°C.
7. Gelatin for water-soluble mounting prepared by dissolving 10 g of gelatin in 60 mL of
distilled water mixed with 250 mg of phenol in 70 mL of glycerol.
8. Poly-
L
-lysine-coated slides: slides are immersed in 0.01% (w/v) poly-
L
-lysine for 5 min
and dried at room temperature.
3. Methods
3.1. Observation and Measurement
of Mucous Thickness on Unfixed Mucosal Sections (
see
Notes 1–4)
This is a rapid and simple method for observing the adherent mucous layer and
measuring its thickness on unfixed sections of mucosa (3). This method has been used to
measure mucous thickness on resected human mucosa both in the stomach (26) and in
the colon (27) or in experimental animal models, particularly rat (3,9,28,29). This
method relies on the adherent gel being sufficiently firm that it is relatively undistorted
when the mucosa is sectioned by parallel razor blades 1.6 mm apart.
1. It is essential that the mucosa and sections are immersed in isotonic saline throughout the
procedure except during sectioning of the mucosa in order to maintain the gel in its fully
hydrated state.
GI Adherent Mucous Gel Barrier 61
2. Gently wash the luminal mucosal surface with isotonic saline (150 mmol/L of NaCl) to
remove food particles and so on as well as nonadhering, soluble mucus.
3. Remove outer muscle layers from serosal side of mucosa by blunt dissection.

4. Mount the mucosa, luminal surface up, on a Millipore filter on a corkboard and cut sec-
tions with a pair of parallel, sharp razor blades separated by a distance of 1.6 mm. The
razor blades are held together with spacers (1.6 mm) between them by bolts tightened by
hand. It is often useful to loosen partially the bolts in order to remove the newly cut
mucosal section from between the razor blades.
5. Position sections transversely on a microscope slide. Check the positioning of the sec-
tions with a low-powered (e.g., × 3.5 ) stereoscopic microscope. Care should be taken not
to distort or stretch the sections during manipulation and positioning.
6. The surface mucous layer can be observed under an inverse microscope (e.g., × 200) using
either light- or dark-field illumination or phase contrast. The adherent mucous layer
appears as a translucent layer between the dense mucosa and the clear bathing solution.
The thickness of the mucous layer is measured using an eyepiece graticule.
7. Individual mucous thickness values should be measured on at least three to six sections
per mucosal specimen. Measurements of gel thickness should be taken at regular inter-
vals along each section; A good distance is 500 µm between each reading (about 6–10
readings per section, depending on its length).
8. Mucous thickness values on unfixed mucosal sections usually show a nonparametric dis-
tribution with more readings at the lower end of the range. Therefore, results are best
treated for statistical significance by methods that compare nonparametric distributions
such as the Mann-Whitney U-test. Values for thickness should therefore be expressed in
terms of medians and upper and lower quartiles.
9. When this technique is used for the first time on a new tissue, it is useful to validate the
method by checking that the thickness of the adherent mucous layer on the sections does
not change significantly over a period of 45 min and that the same mean values for mucous
thickness are obtained for individual sections when read from either side (sections turned
over 180° and remounted).
3.2. Histological Fixation and Staining of Adherent Mucous Gel
on Cryostat Sections of Mucosa (
see
Notes 5–9)

Jordan et al.’s (10) method preserves the mucous layer on cryostat sections of
resected mucosa or biopsies without apparently causing shrinkage and retaining its
original thickness in situ. The cryostat sections undergo no prefixation or long ethanol
dehydration steps characteristic of previous methods. Instead, the tissue is given a
brief ethanol pretreatment followed by a prolonged aqueous staining, and a gentle
postfixation in paraformaldehyde vapor (37°C for 45 min), and is finally mounted in a
water-soluble gelatin gel.
3.2.1. Preparation of Sections
Mucosal segments are snap-frozen in liquid nitrogen and stored at –20°C. For
smaller gut segments, e.g., rat, a good method is to leave the stomach and intestine
intact, ligate one end, fill with 0.9% NaCl, and ligate the other end before snap-freez-
ing. This ensures minimum disturbance of the mucous layers and prevents contact of
the adjacent luminal walls of the tissue. Biopsies, e.g., from human endoscopy
(e.g., 2 mm
2
) need support, which can be provided by wrapping them in a 1–cm
2
sand-