IMMUNO-HISTO-CHEMISTRY

Introduction:

Immunohistochemistry (IHC) refers to the process of detecting antigens in cells of a tissue section by using principle of antigen-antibody reactions. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristics of particular cellular events such as proliferation or apoptosis. IHC is widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.

Albert Coons conceptualized and first implement the procedure in 1941

Visualizing and antigen-antibody reaction can be accomplished in a number of ways. Mainly either of the following:

1.       Chromogenic Immunohistochemistry (CIHC): An antibody is conjugated to an enzyme, that can catalyse a colour producing reaction.

2.       Immunofluorescence: The antibody is tagged to a fluorophore such as fluorescein or rhodamine.

Purpose:

1.       IHC can be used to detect mutations in genes that are associated with the development of breast cancer and the risk of developing breast cancer.

2.       IHC can also detect HER2 hormone receptor on the tumor surface. Which rates in on a scale from 0-3⁺.

3.       IHC used to identify replicating cells. Locate cells that are signalling and also helps to locate apoptic cells.

4.       IHC identify the different types of cells in a tissue and detect cellular antigen. Also, it examines the cytoskeletal structure.

Standard IHC Staining Procedure:

1.       3μm Thick sections are mounted on slides coated with adhesives and fixed in incubator for overnight at 37°C or incubated at 60°C for 1hr.

2.       Deparaffinize sections in xylene and grades of alcohol to water.

3.       Quench endogenous peroxidase activity. Incubate slide in hydrogen peroxide block for 10 minutes.

4.       Rinse in buffer wash foe 5 minutes.

5.       Perform antigen retrieval step as desired either using enzymes or heat mediated protocols.

6.       Rinse in buffer wash for % minutes.

7.       Incubate with primary “blocking serum” for 5 minutes.

8.       Rinse buffer wash. This step can be optional.

9.       Incubate with primary antibody for 1hr at room temperature.

10.   Rinse in buffer wash for 10 minutes. (2 changes of 5 min each)

11.   Incubate with secondary link antibody for 20 to 30 minutes

12.   Rinse in buffer wash for 10 minutes.

13.   Apply chromogen substrate for 5 to 10 minutes.

14.   Rinse in distilled water for 5 minutes.

15.   Counter stain with hematoxylin.

16.   Rinse in running tap water.

17.   Dehydrate, clean and coverslip using DPX.

Staining protocol for frozen section:

1.       Snap freeze small pieces of tissue.

2.       Cut frozen sections at 4 microns.

3.       Pic up sections on gelatine chrome coated or PLL coated slide.

4.       Airdry overnight at room temperature (25°C -30°C)

5.       Fix in cold acetone for 20 minutes.

6.       Air dry sections.

7.       Proceed with immune-staining.

For frozen section endogenous peroxidase blocking is not recommended but the use of negative control for the identification of endogenous peroxidase is preferred.

       

Immunohistochemistry Staining Procedure:

Sample Preparation: Proper preparation of the sample is critical to maintain cell morphology,
tissue architecture and the antigenicity of target epitopes, depending on the purpose and the objective, thickness of the tissue is determined. About 4-40μm slicing of tissue is obtain by microtome and mounting on slides. After preparation of sample or slices, the slices deparaffinize in xylene and ascending grades of alcohol to water. Due to fixation and specimen preservation, additional steps are required to make the specific cell epitopes available for antibody binding. Similarly depending on the tissue type and the method of antigen detection endogenous enzymes may need to be blocked.

Selection of paraffin Block: After ample fixation we need to prepare paraffin block of the sample. We use sometime frozen section prepared by cryostat or any other mechanism. Depending on the tissue. We use various temperature for preparation of paraffin block.

Sectioning and Deparaffinization: After the preparation of paraffin block or frozen section, with the help of microtome tissue sectioning is done and then the deparaffinization process of tissue by xylene and ascending grades of alcohol to water. After that the deparaffinized tissue is induced to perform further procedure of IHC that is blocking of endogenous enzymes.

Blocking of Endogenous Enzymes: Before the deparaffinization process we should mount the slide with adhesives poly-l-lysine in an incubator for overnight at 37°C. The next method for blocking of endogenous enzymes we should quench endogenous peroxidase activity and incubate the slide in hydrogen peroxide block for 10 minutes

Antigen Retrieval: Formaldehyde is a commonly used fixative. However, it causes protein linkages, which may result in masking of tissue antigens. Antigen retrieval is pre-treatment prior to immuno-staining used to unmask the antigen. The technique involves use of proteolytic enzyme digestion of heat induced antigen retrieval method.

Primary Antibody: after antigen retrieval incubate the sample with blocking serum for 5 minutes. A respective primary antibody should be added against the respective antigen. This primary antibody is used as a monoclonal antibody which is able to detect only 1 antigen. The antigen is incubated with primary antibody for one hour.

Secondary Antibody: Secondary antibody is mainly used in indirect ELISA test. In this test secondary antibodies provide structural support. Due to the structural bond between 1° and 2° antibody, the washing agent unable to break down the bond between them. A enzyme (HRP) labelled with secondary antibody incubate for 20-30 minutes.

Substrate Chromogen System: A TMB substrate is added to the sample which reacts with Horse reddish Peroxidase (HRP) enzyme and gives an instant colour in an alkaline medium. The intensity of the colour is directly proportional to the concentration of the antigen present in the sample. Apply chromogen substrate for 5-10 minutes.

Counter Staining: After chromogen substrate system a second chemical stain is other applied to provide contrast that helps the primary staining product stand out. The most used counterstain in IHC is Dab, Hematoxylin & Eosin (H&E), Thermo-scientific pierce Hoechst fluorescent stain etc.

Observation: After staining procedure the slide is mounted by DPX and Canada Balsam, therefore the slide observes under the electric microscope at 100x by using cedar wood oil for better visualization.

 

Diagnostic Immunohistochemistry Markers:

Immunohistochemistry (IHC) is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. It is also an effective way to examine the tissue. IHC has become a widely used technique in the neurosciences, enabling researches to examine protein expressions within specific brain structures. Its major disadvantage is that, unlike immunoblotting technique where staining is checked against a molecular weight ladder it is possible to show in IHC that the staining is corresponded with the antigen of interest. For this reason, the primary antibodies must be well validated in a western blot on similar procedure. IHC technique is more widely used in diagnostic pathology for typing tumours, e.g., Immunostaining for e-cadherin (adhesive molecule) to differentiate between DCIS (ductal carcinoma in situ, stains positive) and LCIS (lobular carcinoma in situ, which does not stain positive).

Most recently IHC have been useful in differential diagnosis of multiple forms of salivary gland, head, neck carcinomas.

Following is some test used in Immunohistochemistry –

1.Identification of B cell lymphomas using CD20

2. Identification of T cell lymphomas using CD3

3. CD45 and CD30 used in Hodgkin’s disease

4.CD117 used for gastrointestinal stromal tumours.

5.CD10(CALLA) used for renal cell carcinoma and acute lymphoblastic leukaemia

6.Prostat specific antigen used for PROSTAT gland.

7.Cytokeratins used for identification of carcinomas.

8.Estrogens and Progesterone staining for tumour identification.

 

Chemical Inhibitors for Immunohistochemistry (IHC):

Tumor biology allows for a number of potential intracellular targets. Many tumors are hormone dependent. The presence of hormone receptors can be used to determine if a tumor is potentially responsive to antimonial therapy. One of the first therapies was the antioestrogen, tamoxifen, used to treat breast cancer such hormone receptors can be detected by IHC. Imatinib, an intracellular tyrosine kinase inhibitor was developed to trear chronic myelogenous lukemia, a disease characterised by the formation of a specific abnormal tyrosine kinase. Imatinib has proven effective in tumors that express other tyrosine kinase. Most notably KIT. Most gastrointestinal stromal tumors express KIT. Which can be deleted by Immunohistochemistry.  

Clinical Significance: 

IHC is a method for detecting antigens or haptens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC used for diagnosis of cancer, Specific tumor antigens are expressed denovo or upregulated in certain cancers.

A positive test means that a marker or receptor is bound on the cell during the biopsy or indicates a certain change in the protein of the tumor.      

The Black-headed and White-headed seagulls in Raichyak

The Black-headed and White-headed seagulls in Raichyak 

Raichak is a small town in South 24 Parganas district of West Bengal, about 50 km away from Kolkata. It is a riverside retreat that offers a serene and scenic escape from the hustle and bustle of the city. Raichak has a rich history and culture, as it was once a fort and a trading post for the British and the Dutch. It also has a variety of attractions and activities for visitors, such as resorts, spas, country homes, lighthouses, temples, and more. But one of the most fascinating features of Raichak is its abundance of seagulls that visit the river Hooghly every winter. These graceful and gregarious birds add a touch of beauty and charm to the landscape and create a memorable experience for anyone who sees them.

Seagulls are a group of birds that belong to the family Laridae, which includes gulls, terns, and skimmers. They are mostly coastal or inland birds that feed on fish, insects, worms, carrion, garbage, and other items. Seagulls are often seen in large, noisy flocks and have a variety of calls and displays.

One of the most common and widespread seagulls is the black-headed seagull (Chroicocephalus ridibundus), which breeds in much of Europe, Asia, and eastern Canada. Despite its name, it does not have a black head, but rather a chocolate-brown one that turns white in winter. It also has a dark red bill and legs, a white body, and gray wings with black tips. It is a small gull, measuring about 38 cm (15 in) in length and weighing about 300 g. 

The black-headed seagull is a migratory bird that winters in warmer regions of Europe, Africa, Asia, and North America. It prefers wetlands, lakes, rivers, marshes, fields, parks, and urban areas as its habitats. It feeds mainly on insects, worms, crustaceans, mollusks, fish, seeds, berries, and human food scraps. It often follows plows and tractors to catch exposed prey.

The black-headed seagull is a colonial breeder that nests on the ground or on low vegetation near water. It builds a cup-shaped nest of grasses, reeds, twigs, feathers, and other materials. It lays two to five eggs that are greenish-brown with dark spots. Both parents incubate the eggs for about 22 days and feed the chicks for another 35 days until they fledge. The chicks are brownish-gray with dark streaks and spots.

The black-headed seagull has a lifespan of about 15 years in the wild. It faces threats from habitat loss, pollution, predation, hunting, and human disturbance. However, it is still a common and widespread species with an estimated global population of 18 million individuals. It is classified as least concern by the IUCN Red List of Threatened Species.

Another seagull that has a white head is the herring gull (Larus argentatus), which breeds in North America, Europe, and Asia. It has a white head that stays white throughout the year. It also has a yellow bill with a red spot near the tip, pink legs, a white body, and gray wings with black tips. It is a large gull, measuring about 60 cm (24 in) in length and weighing about 1 kg (2.2 lb).

The herring gull is also a migratory bird that winters along coasts or inland waters of temperate or subtropical regions. It prefers marine habitats such as cliffs, islands, beaches, harbors, estuaries, and offshore waters. It feeds mainly on fish, crustaceans, mollusks, worms, insects, birds' eggs and chicks, carrion,

garbage,and other items. It often scavenges from fishing boats or dumps.

The herring gull is also a colonial breeder that nests on cliffs or islands near water. It builds a large nest of grasses,

twigs,

seaweed,

and other materials. It lays two to four eggs that are greenish-brown with dark blotches. Both parents incubate the eggs for about 28 days and feed the chicks for another 50 days until they fledge. The chicks are grayish-brown with dark spots.The herring gull has a lifespan of about 20 years in the wild. It faces threats from habitat loss,

pollution,

predation,

hunting,

and human disturbance. However,

it is still a common and widespread species with an estimated global population of 10 million individuals. It is classified as least concern by the IUCN Red List of Threatened Species.

Black-headed seagulls and herring gulls are two examples of seagulls that have different head colors during different seasons or throughout the year. They are both adaptable and opportunistic birds that can live in various habitats and feed on various foods. They are both important members of the ecosystem that provide services such as scavenging,

pest control,

and seed dispersal.

One of the most enjoyable ways to see the seagulls in Raichak is to take a ferry ride across the river Hooghly. The ferries operate between different towns and cities along the river, such as Howrah, Kolkata, Diamond Harbour, Chandannagar, and Chinsurah. As the ferry moves along the water, hundreds of seagulls follow it and fly around it, creating a spectacular sight. The seagulls are not shy and often come close to the ferry passengers, who feed them with snacks and bread. The seagulls catch the food in mid-air or dive into the water to retrieve it. The feeding frenzy attracts more seagulls and makes them more playful and lively. The ferry passengers enjoy watching and interacting with the seagulls, who add fun and excitement to their journey.

International Beaver Day

 International Beaver Day: 7th April 

International Beaver Day is celebrated every year on April 7. It was created by Beavers Wetlands & Wildlife (B.W.W.), a non-profit organization in the United States, Canada, and other countries in 2009. The beaver is a special and important animal that plays a significant role in the ecosystem. B.W.W. has been working hard since 1985 to raise awareness about beavers through research and education.

Beavers are semi-aquatic animals that live in places like Canada, Poland, Germany, France, central Russia, and southern Scandinavia. They like to live in freshwater areas like rivers, lakes, streams, and ponds. Beavers have big heads, stout bodies, brown or grey fur, webbed front feet, and hand-like front feet. Their flat, scaly tail is their most distinctive feature, which they use for communication and balance.

Beavers are a keystone species, which means they have a large impact on the environment in proportion to their population. They eat trees and use the leftover wood to build dams and lodges. They also move mud, rocks, and debris around to create their homes. Beavers were once hunted for their fur and meat and almost became extinct. There used to be around 60 million beavers in North America, but their population has decreased to between 10 and 15 million.

International Beaver Day is celebrated by visiting a beaver pond to observe them, creating funny posts about beavers, and sharing information about them on social media with the hashtag #internationalbeaverday.

Here are five surprising facts about beavers:

Canada’s national animal is the beaver, the Catholic Church once declared beavers to be fish for Lent, beavers are typically monogamous and can stay together for multiple breeding seasons, beavers work at night to build their homes, and beavers have transparent eyelids that help them see underwater.

Certainly! Beavers are remarkable animals that have a significant impact on the ecosystems in which they live. They are known for their dam-building abilities, which create wetlands that serve as important habitats for a wide range of plants and animals. These wetlands help to filter water, prevent erosion, and regulate stream flow, making them essential for the health of many ecosystems.

Beavers are also fascinating creatures to observe. They are semiaquatic, which means they spend a lot of time in the water, and their adaptations for this lifestyle are impressive. For example, they have webbed feet that help them to swim and dive, and they can hold their breath for up to 15 minutes underwater.

Despite their importance, beavers have faced many challenges over the years. They were once hunted extensively for their fur and meat, which nearly led to their extinction. However, conservation efforts have helped to stabilize their populations, and they are now found in many parts of the Northern Hemisphere.

Overall, beavers are a wonderful reminder of the important role that all species play in maintaining healthy ecosystems, and International Beaver Day is a great opportunity to celebrate these amazing animals and raise awareness about the need to protect them and their habitats.

Abortion and its Related Issues

Abortion and its Related Issues :

 Abortion is a highly debated topic that has been controversial for many years. Despite the differences in opinion, it is essential to understand the different viewpoints and considerations that come with this issue. This article aims to provide a detailed analysis of the moral, ethical, practical, and health-related aspects of abortion.

Background :

Abortion refers to the termination of a pregnancy, either spontaneously or intentionally, before the fetus can survive outside the womb. The procedure can be performed for various reasons, including medical conditions that endanger the mother's life, fetal abnormalities, or personal choice. Depending on the stage of pregnancy, there are different methods of abortion, such as medication-induced abortion or surgical abortion.

The legality of abortion varies across different countries and regions. Some countries have strict laws against abortion, while others allow it under certain circumstances, such as in cases of rape or incest, fetal abnormality, or when the mother's life is at risk. In some countries, abortion is legal and widely available on request.

Moral and Ethical Considerations :

The morality and ethics of abortion have been contentious issues for many years. Proponents of abortion argue that it is a fundamental human right that allows women to have control over their bodies and reproductive choices. They believe that women should have the right to choose whether or not to continue with a pregnancy, and that denying this right is a violation of their autonomy and bodily integrity.

However, opponents of abortion argue that it is morally wrong and should be prohibited. They argue that the fetus has a right to life, and that abortion is equivalent to killing an innocent human being. They also believe that adoption is a viable alternative to abortion and that women who cannot care for their children should carry their pregnancies to term and give the child up for adoption.

Both sides have valid arguments and reasons for their stance. The decision to have an abortion is deeply personal and complex. It is essential to respect the autonomy and choice of the pregnant woman while recognizing the importance of the fetus's right to life.

Practical Considerations :

In addition to moral and ethical concerns, practical considerations also play a significant role in the abortion debate. Unsafe abortion procedures can lead to serious complications and even death, particularly in countries where abortion is illegal or heavily restricted. Access to safe and legal abortion services is, therefore, a crucial factor in protecting the health and well-being of women and ensuring that they can make informed choices about their reproductive health.

The availability of family planning and reproductive health services is also an essential factor in reducing the need for abortion. Women who have access to contraception and other forms of family planning are less likely to have unintended pregnancies, which may lead to fewer abortions.

Health-Related Considerations :

Abortion has both physical and emotional effects on women. Physical effects may include bleeding, cramping, infection, or damage to reproductive organs. Emotional effects may include guilt, anxiety, depression, and post-traumatic stress disorder (PTSD). However, the impact of abortion on women's health is often overstated, and many women who have had abortions experience few or no negative effects.

The risks associated with abortion are typically lower than those associated with childbirth. However, the risk of complications increases with the gestational age of the fetus and the method used for the abortion. Therefore, it is crucial to have access to safe and legal abortion services that are performed by qualified healthcare providers.

Global Abortion Statistics:

Abortion is a common procedure worldwide, and the rate of abortion varies across different regions and countries. According to the World Health Organization, an estimated 56 million induced abortions occurred globally in 2010, with 87% of these taking place in developing countries. While abortion rates have declined in many developed countries in recent years, they remain high in many developing countries where access to family planning and reproductive health services is limited.

One of the key issues in the abortion debate is the moral status of the fetus. Some argue that a fetus is a person with a right to life from the moment of conception, and that abortion is therefore morally equivalent to murder. Others argue that the fetus is not a person with full moral status until later in pregnancy, or even until birth, and that women should have the right to terminate their pregnancies for any reason.

The philosophical debate over the moral status of the fetus is complex and multifaceted and has been the subject of extensive discussion and debate among scholars and activists. Some argue that the fetus should be considered a person from the moment of conception because it has the potential to become a person, while others argue that potentiality alone is not sufficient to confer full moral status.

Another key issue in the abortion debate is the right to autonomy and bodily integrity. Pro-choice advocates argue that women have the right to control their own bodies and make decisions about their reproductive health, including the decision to have an abortion. They argue that denying women this right is a violation of their autonomy and bodily integrity and that women must be allowed to make their own decisions about their lives and bodies.

Pro-life advocates, on the other hand, argue that the right to life of the fetus trumps the right to autonomy and bodily integrity of the woman. They argue that the fetus is a human being with the right to life, and that abortion is therefore an unjustified taking of innocent life.

A related issue is the question of whether the state has the right to intervene in the reproductive decisions of women. Pro-choice advocates argue that the state has no right to restrict women's access to abortion and that women must be allowed to make their own decisions about their bodies and lives. They also argue that restricting access to abortion can lead to unsafe and illegal abortions, which can put women's lives at risk.

Pro-life advocates, on the other hand, argue that the state has a duty to protect the lives of unborn children and that restricting access to abortion is therefore a legitimate exercise of state power. They also argue that the state has a legitimate interest in promoting the value of human life, and that restricting access to abortion can help to reinforce this value.

One of the practical considerations in the abortion debate is the question of how to provide safe and legal access to abortion services. In many countries, access to abortion is heavily restricted or even illegal, which can lead to unsafe and illegal abortions. This can result in serious health risks for women, as well as social and economic costs.

Pro-choice advocates argue that safe and legal access to abortion is essential for protecting the health and well-being of women, and that restrictions on access to abortion should be lifted. They also argue that access to comprehensive reproductive health services, including contraception, education, and counseling, can help to reduce the need for abortion and promote healthy and sustainable communities.

Pro-life advocates, on the other hand, argue that promoting alternatives to abortion, such as adoption and support for pregnant women, is a more effective way to reduce the demand for abortion. They also argue that access to contraception can promote promiscuity and undermine traditional family values.

In recent years, there has been increasing attention to the intersection of abortion rights and social justice. Some argue that restricting access to abortion disproportionately affects low-income women, women of color, and women in developing countries and that access to safe and legal abortion is therefore a matter of social justice.

Others argue that abortion itself is a social justice issue, as it allows women to control their own bodies and make decisions about their lives and futures. They argue that denying women access to abortion is a form of oppression that perpetuates gender inequality and denies women their fundamental human rights.

In conclusion, abortion is a complex and controversial issue that elicits strong emotions and beliefs. While different individuals, groups, and societies may hold divergent views on the morality, legality, and ethics of abortion, it is important to recognize its significance as a fundamental human right and public health issue. Access to safe and legal abortion services is critical in protecting the health, well-being, and autonomy of women, particularly those in developing countries who may face significant barriers in accessing reproductive health care.

Estimation of Sodium and Potassium in Blood: 

1.  Estimation of Sodium in Blood:

 

§  Method:

§  Principle: The method is based on the reaction of sodium with a chromogen producing a chromophore whose absorbance is directly proportional to sodium concentration in the test specimen which can be photometrically measured.

§  Procedure:

	Test	Standard	Blank
Reagent	1000μl	1000μl	1000μl
Standard		10μl
Sample	10μl

 

§  Requirement: i. Sodium reagent

ii. Test-tube

iii. 1.0 Cm cuvette

iv. General lab equipment

v. Colorimeter or Semiauto-analyser

§  Incubation: Incubated the assay mixture for 5min at room temperature. Measured the absorbance of the standard and sample against the reagent blank at 630nm.

 

§ Calculation: Sodium in (mEq/L) = OD of Sample / OD of Stand. x Conc. Of Standard (150)

 

§  Linearity: This produce is linear up to 180mEq/L. If values exceed this limit, we should dilute the sample with distilled water and multiply the results with the proper dilution factor.

 

§  Normal Value: In serum/Plasma – 135-155mEq/L.

 

§  Clinical Significance: Sodium is the major cation of extra-cellular fluid. It plays a central role in the maintenance of the normal distribution of water and the osmotic pressure in the various fluid. The main source of body sodium is sodium chloride contained in ingested foods. Only about 1/3^rd of the total body’s sodium is contained in the skeleton since most of its present in the extra-cellular body fluids.

22.  Estimation of Potassium in Blood:

 

§  Method: Turbidimetric method.

§  Principle: The amount of potassium is determined by using sodium tera phenyl born in a specifically prepared mixture to produce a colloidal suspension. The turbidity of which is proportional to potassium concentration in the range of 2-7 mEq/L. Tetra phenyl boron+k+ ‘White Turbidity’. The amount of the turbidity produce is directly proportional to the concentration of potassium in the sample.

 

NaB(C6H5)4 + K+   ------------>   KB(C6H5)4 (White precipitate of potassium tetraphenyl)

 

§  Procedure:

	Test	Standard	Blank
Reagent	1000μl	1000μl	1000μl
Standard		20μl
Sample	20μl

 

§  Requirement: i. Potassium Reagent

ii.  Test-tube

iii. Cuvette

iv. General lab equipment

v. Colorimeter or semiauto analyser

§  Incubation: We will mix the mixture well and incubate the assay mixture for 5mins at room temperature and then we measured the absorbance of the standard and sample against the reagent blank at 630nm.

§  Calculation: Potassium in mEq/L = OD of Sample / OD of Stand x Conc. Of Standard (5)

§  Linearity: This procedure is linear upto 7 mEq/L. If the values exceed his limit, then we should dilute the sample with distilled water and multiply the results with proper dilution factor.

§  Normal Value: Serum/Plasma – 3.5-5.5 mEq/L.

§  Clinical Significance: Potassium is estimated by the turbidimetric method. Potassium ions in the specimen react with sodium tetraphenyl boron to produce an insoluble potassium tetraphenyl boron resulting in a turbid suspension.