Comparison of Staining Reaction of Periodic Acid Schiff with Unripe Orange Extract-Schiff on Skin and Intestinal Tissues

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Okorie Nnaemeka
N.K.S.T. Len Gebrielse’ School of Medical Laboratory Science Mkar, Benue State, Nigeria.
Fasogbon Samuel Ayobami
Public Health In-vitro Diagnostic Control Laboratory, Medical Laboratory Science Council of Nigeria, Lagos State, Nigeria
Adeluwoye Adekunle Oluwatosin
Department of Medical Laboratory Science, Lead City University, Ibadan, Oyo State.
Ajileye Ayodeji Blessing
Department of Medical Laboratory Science, College of Medicine and Health Sciences, AfeBabalola University, Ado Ekiti, Ekiti State.

ABSTRACT
The aim of this study is to compare thehistochemicalreaction of unripe orange extract juice-Schiff with that of Periodic acid Schiff reagent on skin and intestinal tissues.Four (4) tissue sections each obtained from a processed skin and intestine of a wistar rat were prepared and used for this study. The unripe orange juice was extracted and the various pH was measured with different timing. The control tissue sections were stained with periodic acid Schiff technique while the test tissue sections were stained with the unripe orange juice extraction obtained replacing the periodic acid solution in the PAS technique.Unripe orange juice (UOJ) which has similar pH with periodic acid Schiff react with the glycogen and mucosubstances on skin and intestinal tissue of the wistar rat used in this study, by staining them purple magenta similar to what is obtainable with periodic acid Schiff (PAS) staining protocol. In conclusion, unripe Orange JuiceSchiff technique has stained the skin and intestine of the Wister rat in similar manner to periodic acid Schiff technique.

Keywords: Histochemical, Periodic acid Schiff, Intestinal tissue, Skin tissue, Mucosubstances, Glycogen.

INTRODUCTION

Periodic Acid Schiff (PAS) is a staining method used to detect polysaccharide such as glycogen, and mucosubstances such as glycoproteins, glycolipid and mucins in tissues. The reaction of periodic acid oxidizes the vicinal diols in these sugars. Usually breaking up the bond between two adjacent carbons not involved in the glycosidic linkage or ring closure in the ring of the monosaccharide unit that are parts of long polysaccharides, and creating a pair of aldehydes at the two free tips of each broken monosaccrharide ring1. The oxidation condition has to be sufficiently regulated so as not tooxidize the aldehydes further. These aldehydes then react with the Schiff reagent to give a purple magenta color. A suitable basic stain is often used as a counter stain2.
The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid of the Schiff solution forming the magenta color3.PAS staining is mainly used for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), typically found in e.g. connective tissues, mucus, the glycocalyx, and basal laminae.PAS diastase stain (PAS-D) is PAS stain used in
combination with diastase, an enzyme that breaks down glycogen. Alcian blue/periodic acid–Schiff (AB/PAS or AB-PAS) uses alcian blue before the PAS step4. Presence of glycogen can be confirmed on a section of tissue by using diastase to digest the glycogen from a section, then comparing a diastase digested PAS section with a normal PAS section. The diastase negative slide will show a magenta staining where glycogen is present within a section of tissue. The slide that has been treated with diastase will lack any positive PAS staining in those locations on the slide.
Among several immense use that has been found for Periodic acid Schiff staining in the diagnosis of medical conditions are glycogen storage disease, adenocarcinomas (which often secrete neutral mucins), paget disease of the breast, Alveolar soft part sarcoma, staining macrophages in Whipple’s disease, Pulmonary alveolar proteinosis, and erythroleukemia (leukemia of immature red blood cells). It can be used to diagnose a1-antitrypsin deficiency if periportal liver hepatocytes stain positive, as well as pulmonary interstitial glycogenosis (PIG), a condition where glycogen is identified in lung biopsy specimens of infants.
Orange is one of the largest citrus grown fruit in Nigeria5 and is one of the main sources of vitamin C (ascorbic acid) in Nigerian diets. Other vitamins that are of dietary significance are found in orange and include folic acid and thiamine.Orange juice also contains niacin, riboflavin and pantothenic acid vitamins although in minute quantities of about 2 to 4%6. Orange as one of the citrus fruits is non-climacteric, hence, they do not ripen or improve in quality after harvest. Various methods which can be used for processing of orange juice include the traditional thermal processing method (pasteurization) which kills bacterial and extend the shelf-life of juices, but affects the taste of the product. The pressurized carbon dioxide process, according to7 showed that this process is as effective as heat pasteurization, but does not change the taste and preserves more of the vitamins found in fresh squeezed juice. The periodic acid Schiff reaction is used to basement membrane, glycogen and neutral acidic mucosubstances will appear purple due to positive reaction with both alcianlue and periodic acid Schiff. According to8 periodic acid stain is useful for many things. It stain glycogen, mucin, mucoprotein as well as glycoprotein and fungi. A pre-digestion step with amylase will remove staining for glycogen periodic acid Schiff is useful for outlining tissue structure basement membranes capsules, blood vessels, etc8.

Orange juice have been found to be of medicinal importance in folkloric medicine. Among uses found for orange juice includes the reduction of the presence and effects of “bad” cholesterol, why increasing the amount of “good” cholesterol in the body, boosting of immune system, as well as dissolution of kidney stone.Orange juice neutralizes the pro inflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin induced toxicity. Similarly, in cancer prevention, since one of the most important functions of antioxidants is to prevent cancer, orange juice is very rich in vitamin C which works as an antioxidant. Antioxidants keep the DNA of healthy cells from mutating into cancerous cells,hence antioxidants like vitamin C are the first line of defense for cancer and other serious diseases9. Having been observed for it acidity level, this study therefore compare the unripe orange juice with periodic acid as a component solution in the Periodic acid Schiff (PAS) staining technique, which is a staining protocol carried out at an acidic pH of 3.9.This is particularly important as naturally occurring dyes from plants are being viewed as an alternative to synthetic dyes for many developing countries that can no longer afford the ever increasing cost of synthetic dyes.

MATERIALS AND METHOD
Ethical Clearance
Ethical approval for this research study was obtained from the research ethics committee of the N.K.S.T. Len Gabrielse School of Medical Laboratory Science, the experiment was carried out in strict accordance with the guidelines for the care and use of animals for research.

Experimental Animal
Wister rat was used for this scientific research. The animal was handled humanely in conformity with the established ethical guideline for the care and use of laboratory animals.

Extraction of orange juice
For thisstudy, unripe orange fruit where obtained freshly from the orange tree stem at Mkar, Gboko Local Government area of Benue State, Nigeria.The unripe orange fruit were peel using knife and were then sliced into smaller pieces. The sliced unripe orange were parked inside the juice extractor machine, in order to extract the juice separating it from the chaff.The juice was filtered using four layers of gauze, and labeled with date.

Procedure for testing pH of unripe orange juice (UOJ)
The pH meter was inserted in the extracted unripe orange juice (UOJ) to measure the pH of the orange juice solution. The result of freshly prepared orange juice was 3.6. After 24 hours, the pH remains 3.6, only measuring 3.4 after 48hours, an indication of reduction due to fermentation.

Study Design
A total number of eight (8) tissue sections, four (4) each from skin and intestine of a wistar rat were prepared and used for this study. Skin and Intestine tissues were selected for this study as they have been histologically demonstrated to be rich tissue source of localized mucosubstances and glycogen.

Preparation of Slide
Four sections were obtained from a processed intestine tissue of the wistar rat and labeled as follow:
IC – Intestine control at pH 3.9
IT1 – Intestine test 1 at pH 3.6(pH of freshly prepared UOJ)
IT2 – Intestine test 2 at pH 3.6(pH after 24 hours of UOJ)
IT3 – Intestine test 3 at pH 3.4(pH after 48 hours of UOJ)
Also, another four tissue sections were obtained from a processed skin tissue of the wistar rat and labeled as follow:
SC – Skin control at pH 3.9
ST1 – Skin test 1 at pH 3.6(pH of freshly prepared UOJ)
ST2 – Skin test 2 at pH 3.6(pH after 24 hours of UOJ)
ST3 – Skin test 3 at pH 3.4(pH after 48 hours of UOJ)

Staining Procedure Using PAS (Control Slides)
i. Control slide IC and SC were dewaxed and hydrated using descending grades of alcohol (100%, 90%, 70%)
ii. They were taken to water
iii. They were oxidized using 1% Periodic acid at pH 3.9 for 10 minutes
iv. Both slides were rinsed with tapwater
v. They were then stained with Schiff reagent for 20 minutes
vi. Slides IC and SC were again rinsed thoroughly in tapwater
vii. They were counterstained with Cole’s haematoxylin for 10 minutes
viii. They were rinsed with tapwater
ix. Differentiated with 1% acid alcohol
x. Rinsed with water
xi. They were blued in tap water for 10 minutes grades of alcohol (70%, 90%, and 100%).
xiii. Cleared using xylene and mounted using Diphthalenexylene (DPX)

Staining Procedure Using Orange Juice Extract (Test Slides)
i. Sections marked IT1, IT2, IT3 and ST1, ST2, ST3 were dewaxed and hydrated in descending grades of alcohol (100%, 90%, 70%)
ii. Sections were taken to water
iii. Slide IT1, IT2, and IT3 were oxidized with unripe orange juice at pH 3.6 (fresh), 3.6(24hrs), and 3.4 respectively for 10 minutes
iv. Slide ST1, ST2, ST3 were oxidized with unripe orange juice at pH 3.6 (fresh), 3.6 (24hrs), and 3.4 respectively for 10 minutes
v. All the sections were rinsed thoroughly in tap water
vi. They were stained with Schiff solution for 20 minutes
vii. Rinsed thoroughly in tap water
viii. They were counterstained with Cole’s haematoxylin for 10 minutes
ix. They were again rinsed in tap water
x. Differentiated briefly with 1% acid alcohol
xi. They were blued in tap water for 10 minutes
xii. Dehydrated using ascending grades of alcohol (70%, 90%, 100%)
xiii. Cleared using xylene and mounted using Diphthalenexylene (DPX)
All slides were examined under the binocular microscope using 10x, 40x and 100x objectives respectively.

RESULTS
Histology of Intestinal Tissue Sections
Histology of Intestinal Control (IC) pH 3.9 micrograph

 

Discussion
Cell structures take up specific stains to varying degrees based on their biochemical characteristics. Many stains which are suited for particular purposes and allowing cell structures to be differentiated have been developed by histologists. The unripe orange juice which have the similar pH with that of periodic acid solution in the Periodic Acid Schiff (PAS) stain, stained clearly, the glycogen components of the intestinal tissue at pH3.6 (both fresh and 24hrs), as well as at pH 3.4. However for the intestinal tissue, mucosubstances (glycogen) were clearlydemonstrated at pH 3.4. Goblet cells of the intestine were also stained by the unripe orange juice due to its positive reaction with Schiff solution. For the skin tissue sections, similar to the PAS technique which serves as control,the unripe orange juice stained the glycogen, stratum adiposum, and hair follicles on skin tissues of wistar rat as indicated in figures 6-8 above. It is of note too that neoplastic cells on the skin test sections, (ST1, ST2, and ST3) were also demonstrated (indicated by yellow arrow).
Conclusion
Based on the findings of this study, it is observed that, unripe orange juice which has similar pH with periodic acid Schiff react with the glycogen and mucosubstances on skin and intestinal tissue of the wistar rat, by staining them purple magenta similar to what is obtainable with periodic acid Schiff (PAS) staining protocol.

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