experimental birds : seven - day - old white leghorn male chickens were used in 
 this study .
the birds were fed a commercial balanced diet and water ad 
 libitum and maintained under controlled light conditions ( 12-hr light/12-hr 
 dark ) . at the end of the experimental period
, the birds were euthanized , and their tissues 
 were used as described below .
the birds were treated in accordance with the guideline for 
 regulation of animal experimentation ( 1997 ) of the faculty of agriculture , shinshu 
 university .
tissue samples : the experimental methods were divided into three kinds as 
 follows : a pre - embedding technique for immunoelectron microscopy reprocessed from paraffin 
 sections stained by the linked streptavidin - biotin method ( prlsb ) that had been coverslipped 
 with entellan ( merck , darmstadt , germany ) and completed light microscopic 
 observation 1 year or more before ; a pre - embedding technique for immunoelectron microscopy 
 reprocessed from paraffin sections that had been newly sliced at a thickness of 5 
 m from previously embedded paraffin blocks and immunohistochemically 
 stained by colloidal - gold immunolabeling ( prcg ) ; and a post - embedding technique for 
 immunoelectron microscopy from sections newly sampled and stained by colloidal - gold 
 immunolabeling ( pocg ) .
paraffin sections in both prlsb and prcg were mounted on mas - gp 
 coated glass microscope slides ( matsunami glass industries ltd . ,
the birds ( n=4 ) used for prlsb and prcg were perfused with physiological salt solution , 
 followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.6 ) .
the distal ileum was 
 immediately dissected out after the perfusion and cut into small blocks with razor blades . 

the tissue blocks were cut open along the mesenterium , stretched on a cork plate , immersed 
 in the same perfusate at 4c overnight , washed several times with phosphate buffer and 
 embedded in paraffin wax according to standard procedures .
the birds ( n=4 ) used for pocg were perfused with 4% paraformaldehyde and 0.01% 
 glutaraldehyde in phosphate - buffered saline ( pbs ) .
the distal ileum was sliced under a 
 dissecting microscope into 1  1  2-mm blocks , immersed in the same perfusate at 4c 
 overnight , dehydrated through a graded series of ethanol and embedded in quetol 812 ( nisshin 
 em , tokyo , japan ) .
the linked streptavidin - biotin staining was visualized with 0.05% 
 3,3-diaminobenzidine ( dab ) as described in our previous paper .
briefly , the following antisera were used : rabbit antiserum against 
 synthetic human glp-1 ( amino acids 119 ) ( 1:2,000 dilution ; ga1176 ; affiniti research 
 products , devon , u.k . ) , which does not cross - react with other proglucagon - derived peptides 
 ; and biotinylated goat anti - rabbit igg serum 
 ( diluted 1:300 ; ap132b ; millipore , billerica , ma , u.s.a . ) .
sections on glass microscope 
 slides were soaked in xylene for 12 hr to remove the coverslips and then rehydrated through 
 a decreasing graded series of ethanol for 5 min each .
after washing with pbs , the sections 
 were examined under a light microscope , and the intestinal mucosa containing glp-1-positive 
 cells was dissected into 1  1-mm pieces .
the sections were immersed in 2.5% glutaraldehyde 
 in 0.1 m phosphate buffer ( ph 7.4 ) for 15 min , postfixed in 1% osmium tetroxide 

( oso4 ) for 15 min , dehydrated through a graded series of ethanol and embedded 
 in quetol 812 .
after polymerization of the resin , the sections were detached from the glass 
 slides by warming .
ultra - thin sections ( 80 nm ) were obtained using an ultramicrotome ( super 
 nova ; reichert - jung , vienna , austria ) , mounted on 200-mesh nickel grids ( nisshin em ) , 
 stained with 10% ( v / v ) ti blue ( nisshin em ) and 2% ( w / v ) lead citrate and observed under a 
 transmission electron microscope ( jem-1400 ; jeol , tokyo , japan ) .
paraffin sections were newly cut at 5 m thickness 
 from blocks embedded 1 year before . after routine dewaxing and rehydration ,
the sections 
 were pretreated with 2.5% normal donkey serum ( ihr-8135 ; immunobioscience , mukilteo , wa , 
 u.s.a . ) for 20 min and incubated with the above - described rabbit antiserum against synthetic 
 glp-1 ( 1:2,000 dilution ) for 12 hr .
after three washes with pbs , the sections were incubated 
 with colloidal gold ( 6-nm diameter)-labeled donkey anti - rabbit igg serum ( 1:100 dilution ; 
 711195 - 152 ; jackson immunoresearch , west grove , pa , u.s.a . ) for 3 hr .
the sections were 
 then immersed in 2.5% glutaraldehyde for 15 min and serially processed for the 
 silver - enhanced colloidal gold method   by 
 immersion in 0.1 m acetic acid buffer ( ph 7.0 ) for 10 min to yield detailed 
 electron - immunoreactive structures and enhancement using a silver enhancement kit ( hq 
 silver ; nanoprobes , yaphank , ny , u.s.a . ) in a dark room for 8 min . after thorough washing 
 with 0.1 m phosphate buffer , dissection , postfixation with oso4 , embedding in 
 quetol 812 and electron microscopic observation were performed as described for prlsb .
ultra - thin sections from blocks embedded in quetol 812 were obtained 
 as described above .
the ultra - thin sections were pretreated with 1% ( w / v ) sodium periodate 
 solution for semi - deresination to readily allow reactions between the antibody and antigen 
 and incubated with 1% normal donkey serum for 15 min .
after three washes with pbs , a mouse 
 monoclonal primary antibody against synthetic human glp-1 ( amino acids 736 ) ( 1:8,000 
 dilution ; a6104.1 ; immundiagnostik , bensheim , germany ) was applied to the sections for 24 
 hr .
after three washes with pbs , the sections were treated with colloidal gold ( 12-nm 
 diameter)-labeled donkey anti - mouse igg serum ( 1:200 dilution ; 715 - 205 - 150 ; jackson 
 immunoresearch ) for 2 hr , stained with 10% ( v / v ) ti blue and 2% ( w / v ) lead citrate and 
 observed under a transmission electron microscope .
the specificity of the primary antibody 
 used in this study has been documented by the manufacturer and in previous reports [ 4 , 12 , 21 ] .
morphometry of glp-1 granules : morphometric analyses were carried out to 
 characterize the secretory granules of l - cells .
l - cells that contained a nucleus and more 
 than 10 granules were selected for morphometric analyses . a total of 471 secretory granules 
 from 15 cells in prlsb , 195 secretory granules from 10 cells in prcg and 207 granules from 
 12 cells in pocg located at the basal or perinuclear region of the cytoplasm were randomly 
 selected .
the shortest and longest diameters of the secretory granules were measured using 
 an image analyzer ( ks 400 ; carl zeiss , oberkochen , germany ) .
differences between the mean 
 diameters of granules from each method were tested by anova and with a selected tukey 
immunoreactive cells for the glp-1 antiserum , so - called l - cells , were observed in the full 
 length of the distal ileum in prlsb and prcg under a light microscope .
the cells were 
 distributed in the epithelium of the crypt and the lower part of the intestinal villi and 
 had comma- or flask - like shapes ( fig .
( a ) a 
 section stained with the prlsb procedure and counterstained with mayer s hematoxylin . 

the immunoreactive cells are 
 distributed in the crypts and lower part of the villous epithelium ( arrows ) .
( a ) distal ileum embedded in paraffin and paraffin sections mounted on a 
 glass slide .
( b ) a section stained with the prcg procedure examined under a light 
 microscope without a coverslip .
the arrow indicates an immunoreactive cell observed 
 under the electron microscope in panel d. ( c ) dissected sections including 
 immunoreactive cells embedded in quetol 812 .
the cell 
 surrounded by a dashed line is the same cell indicated by the arrow in panel b. 
 secretory granules accumulate in the basal cytoplasm of l - cells ( asterisk ) .
. shows results of the prcg procedure from a paraffin block to electron microscopic 
 observation .
( a ) a 
 section stained with the prlsb procedure and counterstained with mayer s hematoxylin . 

the immunoreactive cells are 
 distributed in the crypts and lower part of the villous epithelium ( arrows ) .
bars=200 
 m . summary of the prcg procedure from a paraffin block to electron microscopic 
 observation .
( a ) distal ileum embedded in paraffin and paraffin sections mounted on a 
 glass slide .
( b ) a section stained with the prcg procedure examined under a light 
 microscope without a coverslip .
the arrow indicates an immunoreactive cell observed 
 under the electron microscope in panel d. ( c ) dissected sections including 
 immunoreactive cells embedded in quetol 812 .
the cell 
 surrounded by a dashed line is the same cell indicated by the arrow in panel b. 
 secretory granules accumulate in the basal cytoplasm of l - cells ( asterisk ) .
the glp-1-containing cells were 
 easily identified as precipitated dark products resulting from dab in prlsb and gold 
 particle enhanced by silver reactions in prcg ( fig .
3.low-magnification electron micrographs of the chicken distal ileum stained with the 
 prlsb ( a ) and prcg ( b ) procedures .
glp-1-positive cells are easily identified at low 
 magnification by the dark dab reaction products and gold particles enhanced by silver 
 reactions ( arrows ) .
an enterochromaffin cell is observed adjacent to the glp-1-positive cell 
 in panel ( b ) . bars=2 m . ) .
on the other hand , it was difficult to identify the granules labeled with the 
 12-nm - diameter gold particles under low magnification in pocg .
although degradation of the 
 nucleoplasm and cytoplasm was observed , secretory granules could be identified in prlsb and 
 prcg ( fig .
these cells contained round 
 to oval secretory granules without a halo that mainly accumulated in the basal cytoplasm or 
 perinuclear region of l - cells ( fig .
4.electron micrographs of glp-1-positive cells in the chicken distal ileum stained with 
 the prlsb ( a ) , prcg ( b ) and pocg ( c ) procedures .
the electron densities of the secretory granules 
 observed with the three different experimental methods are medium and almost the same 
 ( arrows ) .
the secretory granules of an enterochromaffin cell adjacent to the 
 glp-1-positive cell show polymorphous shapes and high - electron density ( asterisk ) ( b ) . 

the post - embedding procedure shows fine preservation of the nucleoplasm and cytoplasm 
 ( c ) .
( d ) high - magnification view of the secretory granules 
 in panel ( a ) .
the dab reaction products precipitate around the secretory granules 
 ( arrows ) and in the cytoplasm ( arrowheads ) .
( e ) high - magnification view of the secretory granules in panel ( b ) .
the particles of 
 enhanced colloidal gold mainly precipitate around the secretory granules ( arrows ) , and 
 a few of them are located in the cytoplasm ( arrowheads ) . the profile of the secretory 
 granules is clearly observed .
( f ) high - magnification view of the secretory granules in 
 panel ( c ) .
almost all of the particles of colloidal gold are diffusely arranged on the 
 secretory granules ( arrows ) , and very few of them are located in the cytoplasm 
 ( arrowheads ) .
the electron densities of the secretory granules observed among the three different 
 experimental methods were medium and almost the same .
4b , the immunopositive granules for glp-1 had lower electron 
 density than the secretory granules of adjacent enterochromaffin cells , which were widely 
 distributed in the gastrointestinal tract and had polymorphous secretory granules .
low - magnification electron micrographs of the chicken distal ileum stained with the 
 prlsb ( a ) and prcg ( b ) procedures .
glp-1-positive cells are easily identified at low 
 magnification by the dark dab reaction products and gold particles enhanced by silver 
 reactions ( arrows ) .
an enterochromaffin cell is observed adjacent to the glp-1-positive cell 
 in panel ( b ) . bars=2 m .
electron micrographs of glp-1-positive cells in the chicken distal ileum stained with 
 the prlsb ( a ) , prcg ( b ) and pocg ( c ) procedures .
the electron densities of the secretory granules 
 observed with the three different experimental methods are medium and almost the same 
 ( arrows ) .
the secretory granules of an enterochromaffin cell adjacent to the 
 glp-1-positive cell show polymorphous shapes and high - electron density ( asterisk ) ( b ) . 

the post - embedding procedure shows fine preservation of the nucleoplasm and cytoplasm 
 ( c ) .
( d ) high - magnification view of the secretory granules 
 in panel ( a ) .
the dab reaction products precipitate around the secretory granules 
 ( arrows ) and in the cytoplasm ( arrowheads ) .
( e ) high - magnification view of the secretory granules in panel ( b ) .
the particles of 
 enhanced colloidal gold mainly precipitate around the secretory granules ( arrows ) , and 
 a few of them are located in the cytoplasm ( arrowheads ) . the profile of the secretory 
 granules is clearly observed .
( f ) high - magnification view of the secretory granules in 
 panel ( c ) .
almost all of the particles of colloidal gold are diffusely arranged on the 
 secretory granules ( arrows ) , and very few of them are located in the cytoplasm 
 ( arrowheads ) .
bars=200 
 nm . examination under higher magnification clarified the differences in the immunoreactivities 
 among the three different experimental methods . in prlsb , the dab reaction products 
 precipitated around secretory granules and within the cytoplasm ( fig .
, the gold particles enhanced by silver products 
 mainly precipitated around secretory granules , and a few were located in the cytoplasm 
 ( fig .
the gold particles were 
 mostly found on secretory granules , and very few of them were located in the cytoplasm 
 ( fig .
the profile of the secretory granules 
 was clearly observed in prcg and pocg , but was difficult to identify in prlsb ( fig .
4d4f ) . because the electron density of the dab 
 reaction product was similar to that of the secretory granules , the deposition of dab 
 reaction products around the whole profile of the granules tended to make the boundary 
 between the granules and cytoplasm ambiguous .
the electron density of the dab reaction 
 product was quite low compared with that of the gold particles ( fig . 4e and 4f ) .
the results of the morphometric analyses of the secretory granules are shown in table 1table 1.shortest and longest diameters of the secretory granules in the different 
 proceduresprocedureshortest diameter ( nm)longest diameter ( nm)prlsb311  70 * 378  80*prcg288  58355  69pocg291  56343
* p<0.01 , 
 significant difference from the other procedures .. the longest and shortest diameters of the secretory granules in prlsb were 
 significantly longer than those in both prcg and pocg .
it is mainly classified into three methods , pre - embedding , 
 post - embedding and non - embedding ( ultra - thin frozen section ) methods .
these three methods 
 each have advantages and disadvantages , and researchers are required to select the most 
 effective method appropriate to the tissues under observation .
pre - embedding 
 immunocytochemical staining has provided successful observations by light microscopy and 
 electron microscopy in the same specimen [ 9 , 16 ] . however , permeabilization treatments are sometimes 
 necessary to obtain optimal labeling of intracellular antigenic sites , particularly with 
 thick sections , such as vibratome sections [ 7 , 9 , 14 ] .
the use of 
 penetration agents , such as enzymes , or detergents , such as triton x-100 , often leads to 
 destruction or disorganization of more labile cytoplasmic structures and frequently gives 
 rise to false - positive staining .
in contrast , 
 post - embedding immunocytochemical staining of ultra - thin sections at the electron 
 microscopic level produces high - resolution localization and good morphological preservation . 

however , epoxy resin blocks a 
 number of antigenic epitopes through interactions with tissue components and forms a 
 hydrophobic polymeric network that prevents the diffusion of water - soluble substances , such 
 as antibodies [ 2 , 6 ] . to expose antigenic sites to the immunoreagents ,
etching of epoxy resin 
 ultra - thin tissue sections with reagents , such as periodic acid or hydrogen peroxide , is 
 necessary [ 2 , 12 , 23 ] . some researchers have taken 
 advantage of hydrophilic embedding media , such as lr white / gold embedding resin , to avoid 
 this problem . in either case ,
post - embedding procedures tend to prevent antibody binding to 
 a sufficient number of antigenic sites , which frequently gives rise to false - negative 
 staining . indeed
, the primary antibody used in 
 prlsb and prcg did not react in pocg in the present study , although the specificity of the 
 primary antibody has been proven .
a non - embedding approach , which uses ultra - thin frozen 
 sections and colloidal gold , can overcome the penetration problem and also preserve the 
 tissue antigenicity [ 9 , 19 ] .
paraffin - embedded tissues have been used for immunohistochemical staining for a long time . 

the medium of paraffin allows tissues to be thinly sliced , typically in the range of 515 
 m .
the specimen is rehydrated through a decreasing graded series of 
 ethanol , which facilitates the penetration of antibodies . in this study
, we attempted to 
 observe 5-m sections that were cut from paraffin blocks and immunostained 
 for observation under an electron microscope .
it has been thought that ultrastructural 
 examination of paraffin - embedded tissues is associated with disruption of membranes , loss of 
 clarity and varying degrees of tissue degradation for reasons , such as series of 
 hydration / dehydration processes . however , some studies have documented that electron 
 microscopic observation produced the opposite result [ 8 , 17 ] .
reported that the ultrastructural features of 
 paraffin - embedded tissues that were reprocessed for electron microscopic observation were 
 quite variable and also pointed out that the fixation , rather than the paraffin embedding , 
 seemed to be most important step that affected the quality of the ultrastructural features . 

moreover , they documented that optimal initial fixation in formalin at an appropriate 
 concentration and ph allowed ultrastructural preservation and that ultrastructural 
 examination of paraffin - embedded tissues could be optimized by avoiding paraffin blocks 
 prepared from poorly fixed tissues and tissues adjacent to areas of necrosis .
some studies 
 have clearly documented that organelles , such as nuclei , desmosomes , sarcomeres , 
 intermediate filaments , tonofilaments and electron - dense granules , can be recognized from 
 reprocessed paraffin - embedded tissues under electron microscopic observation , although 
 mitochondria , polyribosomes , microtubules , lipids and glycogen can exhibit various degrees 
 of degradation [ 8 , 22 , 24 , 26 ] .
the above - mentioned studies on reprocessed paraffin - embedded tissues were 
 performed on newly cut sections from paraffin - embedded blocks , but not on already sliced 
 paraffin sections attached to the glass .
to our knowledge , a study about the possibility of 
 observing hormone - containing endocrine cells reprocessed from paraffin - embedded tissues has 
 not been documented .
in the present study , we investigated the possibility not only of 
 endocrine cells in the conventional paraffin section with pre - embedding immunocytochemical 
 electron microscopic observation but also from newly sliced paraffin sections that were 
 subjected to immunostaining .
we observed secretory granules containing chicken glp-1 using 
 prlsb and prcg techniques . at the electron microscopic level
, the immunopositive secretory granules in prlsb and prcg 
 could be recognized more easily than those in pocg under low magnification , owing to the 
 precipitated dark products resulting from dab and gold particles enhanced by silver 
 reactions .
incubations with antisera and other reagents were performed before the ultra - thin 
 sectioning in prlsb and prcg .
in addition , the incubations were performed after the 
 ultra - thin sectioning in pocg .
immunoreaction sites were identified around the secretory 
 granules in prlsb and prcg and on the content of the granules in pocg .
although the granules 
 had the same shape among the three procedures , both the longest and shortest diameters of 
 the secretory granules were significantly longer in prlsb than in both prcg and pocg .
the 
 electron density of the dab reaction product was similar to that of the secretory granules 
 in prlsb , which made the boundary of the granules ambiguous .
baskin et al . 
  suggested that osmium - dab interactions resulted 
 in background problems .
because oso4 is well known as a post - fixative solution , 
 it was used in prlsb and prcg .
we presumed that this use led to the result that the diameter 
 of the measured granules tended to be longer in prlsb .
conversely , the gold particles enhanced by silver products mainly precipitated around 
 secretory granules , and a few of them were located in the cytoplasm in prcg .
since the 
 electron density of the silver - enhanced gold particles was much higher than that of the 
 granules , the boundary between the granules and the immunogold labeling was clearly 
 recognized under both low and high magnifications .
it was presumed that this clarity 
 provided similar evaluation of the granule diameters between prcg and pocg . because of the 
 difficulty in obtaining sagittally sectioned electron micrographs of l - cells that contained 
 many granules , the measured numbers of granules differed among the three groups .
this study 
 is the first to identify the ultrastructure of secretory granules containing an amino acid 
 peptide ( glp-1 ) obtained from paraffin - embedded and sliced sections with similar evaluation 
 results to pocg , which is a commonly used method for post - embedding immunocytochemical 
 studies .
the reprocessing method from thinly sliced paraffin sections in the present study 
 has limitations compared with reprocessing from paraffin blocks .
the reprocessing method 
 from paraffin blocks , in which paraffin - embedded tissues are dewaxed in xylene , hydrated , 
 postfixed , dehydrated and embedded in epoxy resin , shows fine preservation of nuclei and 
 many cytoplasmic organelles [ 8 , 13 , 22 , 24 , 26 ] .
we presumed that this resulted from removal of the coverslip and 
 repeated hydration / dehydration procedures .
however , prcg provided satisfactory information 
 for the secretory granules containing the amino acid peptide hormone .
the fixative solution 
 used in pocg contained glutaraldehyde , which is commonly used for electron microscopic 
 studies and preserves the fine structure of nuclei and many cytoplasmic organelles .
we 
 presumed that the fine preservation in pocg was also caused by glutaraldehyde . to the best of our knowledge
, the present study is the first to report the advantages of 
 the reprocessing method with sliced paraffin sections , which not only provides information 
 on secretory granules containing an amino acid peptide , but is also inexpensive , does not 
 require special devices and can effectively use precious samples that are already 
 paraffin - embedded and unable to be obtained twice , such as the case for endangered animals 
 and rare pathological tissues . in addition , this method may contribute to ethical ideas that 
 have led to a required reduction in the number of laboratory animals used by researchers .
2 , 
 is a useful procedure from the following viewpoints : 1 ) the thinly sliced and hydrated 
 sections allow penetration of antibodies ; 2 ) the gold enhanced by silver provides fine 
 information of immunoreactive cells at the light microscopic level ; and 3 ) the investigator 
 can observe the same cell under a light microscope and the electron microscope .
 abstractthe purpose of the present study was to investigate the possibility of 
 immunoelectron microscopic observation of endocrine cells in paraffin - embedded tissues . 
 
 the procedure , which involves reprocessing from sliced tissues and immunohistochemical 
 staining by colloidal - gold immunolabeling of paraffin sections from paraffin blocks , was 
 able to reveal the fine characteristics of secretory granules containing glucagon - like 
 peptide-1 . 
 morphometric analyses of the secretory granules showed no significant 
 differences between the reprocessing procedure and a conventional post - embedding 
 procedure , which was performed as a control . 
 the reprocessing procedure has some 
 advantages besides providing information on secretory granules containing the amino acid 
 peptide . 
 for example , the same cell can be observed under both a light microscope and the 
 electron microscope . 
 in addition , the high - electron densities of silver - enhanced gold 
 particles are easily recognized , and the boundary between the profile of the granules and 
 the immunogold labeling is clearly shown at the electron microscopic level . furthermore , 
 the procedure , which is inexpensive and does not require special devices , can effectively 
 use precious samples that are already paraffin - embedded and unable to be obtained twice , 
 such as the case for endangered animals and rare pathological tissues . to the best of our 
 knowledge , 
 the present study is the first to report the advantages of the reprocessing 
 method for sliced paraffin sections of gut endocrine cells . 