The primary antibody and the secondary antibody are not compatible. Not enough primary antibody is bound to the protein of interest

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2 Trubleshting and using cntrls in IHC and ICC N staining The primary antibdy and the secndary antibdy are nt cmpatible Use a secndary antibdy that was raised against the species in which the primary was raised (e.g. primary is raised in rabbit, use anti-rabbit secndary). Be sure that the istypes f the primary and secndary are cmpatible (e.g. IgY vs IgG). Nt enugh primary antibdy is bund t the prtein f interest Cncentrate the antibdy mre, incubate lnger (e.g. vernight) at 4 C. The antibdy may nt be suitable fr IHC prcedures which reveal the prtein in its native (3D frm) Check the antibdy datasheet t see if it has been validated in IHC, and what type f IHC (frmalin/pfa fixatin, fresh frzen, etc.). Test the antibdy in a native (nn-denatured) WB t make sure it is nt damaged. The primary/secndary antibdy/amplificatin kit may have lst its activity due t imprper strage, imprper dilutin r extensive freezing/thawing Run psitive cntrls t ensure that the primary/secndary antibdy is wrking prperly (see sectin 4 fr mre infrmatin abut cntrls). The prtein is nt present in the tissue f interest Run a psitive cntrl recmmended by the supplier f the antibdy (see sectin 4 fr mre infrmatin abut cntrls). The prtein f interest is nt abundantly present in the tissue Use an amplificatin step t maximize the signal. Fr example, use a bitin cnjugated secndary antibdy and a cnjugated streptavidin. The secndary antibdy was nt stred in the dark (if yur detectin system is immunflurescence). Always prevent the secndary antibdy frm expsure t light. Deparaffinizatin may be insufficient Deparaffinize sectins lnger and use fresh xylene. 2

3 Fixatin prcedures (using frmalin and parafrmaldehyde fixatives) may be mdifying the epitpe the antibdy recgnizes Use different antigen retrieval methds t unmask the epitpe (heat mediated with ph 6 r ph 9 buffer, enzymatic, etc.). Fix the sectins fr less time. The prtein is lcated in the nucleus and the antibdy (nuclear prtein) cannt penetrate the nucleus Add a strng permeabilizing agent like Tritn X t the blcking buffer and antibdy dilutin buffer. See ur prtcl abut permeabilizatin techniques. The PBS buffer is cntaminated with bacteria that damage the phsphate grups n the prtein f interest Add 0.01% azide in the PBS antibdy strage buffer r use fresh sterile PBS. High backgrund Blcking f nn-specific binding might be absent r insufficient Increase the blcking incubatin perid and cnsider changing blcking agent. Abcam recmmends 10% nrmal serum f the species f the secndary antibdy fr 1hr, r 1-5% BSA fr 30 min fr cells in culture. Anther ptin is t try a secndary antibdy that has been pre-adsrbed against the Ig f the species f yur samples. The primary antibdy cncentratin may be t high Titrate the antibdy t the ptimal cncentratin, dilute the antibdy further and incubate at 4 C (a slw but targeted binding is best). Incubatin temperature may be t high Incubate sectins r cells at 4 C. The secndary antibdy may be binding nn-specifically Run a secndary cntrl withut primary antibdy. If yu see staining with yur secndary nly, change yur secndary r use a secndary antibdy that has been pre-adsrbed against the Ig f the species f yur samples. Tissue nt washed enugh, fixative still present Wash extensively in PBS between all steps. Endgenus perxidases are active 3 Use enzyme inhibitrs i.e. Levamisle (2 mm) fr alkaline phsphatase r H2O2 (0.3% v/v) fr perxidase.

4 Fixatin prcedures (using frmalin r parafrmaldehyde fixatives) are causing autflurescence (if yur detectin system is immunflurescence). Frmalin/PFA usually autflurescence in the green spectrum, s try a flurphre in the red range. Use a flurphre in the infrared range if yu have an infrared detectin system. T much amplificatin (amplificatin technique) Reduce amplificatin incubatin time and dilute the secndary antibdy r amplificatin kit. T much substrate was applied (enzymatic detectin) Dilute substrate mre and reduce substrate incubatin time. The chrmgen reacts with the PBS present in the cells/tissue (enzymatic detectin) Use Tris buffer t wash sectins prir t incubating with the substrate, then wash sectins/cells in Tris buffer. Permeabilizatin has damaged the membrane and remved the membrane prtein (membrane prtein) Use a less stringent detergent (e.g. Tween 20 instead f Tritn X). Or simply remve permeabilizing agent frm yur buffers. See ur prtcl abut permeabilizatin techniques. Nn-specific staining Primary/secndary antibdy cncentratin may be t high Try decreasing the antibdy cncentratin and/r the incubatin perid. Cmpare signal intensity against cells r tissue that d nt express the target. Endgenus perxidases are active Use enzyme inhibitrs i.e. Levamisle (2 mm) fr alkaline phsphatase r H2O2 (0.3% v/v) fr perxidase. The primary antibdy is raised against the same species as the tissue stained (e.g. muse primary antibdy tested n muse tissue). When the secndary antibdy is applied it binds t all the tissue as it is raised against that species Use a primary antibdy raised against a different species than yur tissue. Use a bitinylated primary antibdy and a cnjugated streptavidin fr the detectin system. The sectins/cells have dried ut Keep sectins/cells at high humidity and d nt let them dry ut. 4

5 Using cntrls Psitive cntrls Why psitive cntrls are necessary: T validate the staining in yur sample, use a psitive cntrl. This is a sectin frm a cell line r tissue knwn t express the prtein yu are detecting. A psitive result frm the psitive cntrl, even if the samples are negative, will indicate the prcedure is ptimized and wrking. It will verify that any negative results are valid. Hw t knw what tissue type r cell line is a suitable psitive cntrl: First check the antibdy datasheet. This will ften prvide a suggested psitive cntrl. Always ensure the tissue r cell line yu use is frm a tested species. If the antibdy datasheet des nt list a psitive cntrl, we recmmend the fllwing in these circumstances: Check t see if there are any Abreviews fr the antibdy. Any tissues, cells r lysates that have been used successfully by these custmers can be cnsidered a suitable psitive cntrl. Try lking at the Swiss-Prt r Omnigene database links n the datasheet. These databases will ften have a list f tissues that the prtein is expressed in. These can als be cnsidered suitable psitive cntrls. Check the GeneCards entry fr the prtein. This will usually prvide yu with relative levels f expressin in varius tissues. Check the Human Prtein Atlas fr the prtein. This has a database f prtein detectin in different tissue types, cancers, and cell lines. Find this at If yu still have difficulty finding a suitable cntrl, we recmmend ding a quick literature search n PubMed t see which tissues and cells express the prtein f interest. Negative cntrls Why negative cntrls are necessary: Use a sectin frm a cell line r tissue sample knwn nt t express the prtein yu are detecting. This is t check fr nn-specific binding and false psitive results. Recmmended negative cntrl tissues are knck dwn (KD) r knck ut (KO) tissue samples. N primary cntrls This is when the primary antibdy is nt added t the sample. This indicates if any nn-specific binding r false psitives may be due t nn-specific binding f the secndary antibdy. Antibdy dilutin buffer cntaining n antibdy is used in place f the primary antibdy slutin at this pint in the prcedure. The secndary antibdy is incubated n the sample in the same way as usual. 5

6 Istype cntrls An istype cntrl is an antibdy f the same istype (IgG2a, IgY, etc.), clnality, cnjugate, and hst species as the primary antibdy that is raised against a mlecule that is nn-existent in the sample yu are using. Usually this is raised against a chemical r a nn-mammalian prtein. Use the same cncentratin (μg/ml) fr the istype cntrl antibdy and the primary antibdy. This will determine the level f backgrund in yur sample. Much like the n-primary cntrl step, yu wuld use this n yur sample instead f the specific primary antibdy. Yu wuld then use yur secndary antibdy as usual. Cntrls fr using a transfected cell line: We recmmend t include an endgenus cntrl if yu are testing a sample f recmbinant prtein. This shuld be an essential part f the experimental plans. There are inherent difficulties with antibdy detectin f recmbinant prteins that need t be cnsidered: Flding f the recmbinant prtein may be different frm the endgenus native frm. This flding may be preventing access f the antibdy t the epitpe. This is particularly the case with tagged prteins. Always ensure tags are placed n the N r C terminal end f the recmbinant prtein. Mst imprtantly, always ensure the recmbinant prtein includes the immungen sequence fr the antibdy yu are using. An endgenus psitive cntrl is imprtant t validate the results, as well as t indicate hw well the reagents (e.g. antibdies) and prcedure are wrking. 6